An anti-interference urine microalbumin assay kit

Abstract

The invention provides an anti-interference urine microalbumin assay kit, the kit comprising reagent R1 and reagent R2; reagent R1 comprises the following components: tris(hydroxymethyl)aminomethane buffer, sodium chloride, EDTA-2Na, anti-interference additive, polyethylene glycol 8000, surfactant, PC300; reagent R2 comprises the following components: tris(hydroxymethyl)aminomethane buffer, Alb antiserum, stabilizer, dispersant, PC300. The kit is a liquid kit with strong stability, high accuracy, wide linear range and anti-interference.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical detection, in particular to an interference-resistant urine microalbumin determination kit and application. Background Art

[0002] In 1982, Viberti et al. found that the total protein in the urine of diabetic patients was within the normal range, while the excretion of urinary albumin increased. They first proposed the concept of urinary microalbumin (ALB). Albumin is one of the important plasma proteins. Under normal circumstances, the molecular weight of albumin is large and cannot cross the glomerular basement membrane. Therefore, the urine of healthy people only contains very low concentrations of albumin, specifically no more than 20 mg per liter of urinary albumin, so it is also called "urine microalbumin test". In the case of disease, the glomerular basement membrane is damaged, causing changes in permeability. Any lesion that can cause increased permeability of the glomerular basement membrane can lead to the excretion of albumin. At this time, albumin can enter the urine, and the concentration of urinary albumin can continue to increase. The most ideal method for measuring urinary albumin is to collect a 24-hour urine sample. Since the excretion of urinary albumin varies to a large extent, an increase in albumin excretion in an unscheduled urine sample (random urine) may be meaningless. It is only of diagnostic value if it increases for 2 to 3 times in a row. Urine microalbumin measurement reflects early kidney disease and kidney damage. Pathological increases are seen in diabetic nephropathy, hypertension, and preeclampsia during pregnancy. Urine microalbumin in the early stage is an early signal and omen of kidney disease. At this time, kidney damage is in a period that can still be reversed. If treated in time, the progression of kidney disease can be stopped or reversed. Urine microalbumin detection can be used as a renal function indicator for systemic or local inflammatory responses, such as early kidney lesions caused by urinary tract infections and other reasons; a predictive indicator for complications of acute pancreatitis; and urine microalbumin can also be tested for those who take medications that affect renal function, so as to facilitate early observation of renal function and take measures as soon as possible.

[0003] At present, the commonly used methods for determining urine microalbumin in China are immunolabeling and immunoturbidimetry, and immunoturbidimetry is more commonly used. Urine microalbumin detection is an immunoturbidimetry method. The test principle is: albumin in urine can undergo agglutination reaction with albumin antibodies in the reagent to form an antigen-antibody complex and a certain turbidity. The turbidity is proportional to the content of antigen (urine microalbumin) in the presence of a certain amount of antibody. The content of microalbumin in the sample is measured by multi-point calibration at a wavelength of 340nm. Anything that affects urine turbidity can affect the results of urine microalbumin determination and cause false positives, such as crystalluria and chyluria. Normal urine contains many crystalline and non-crystalline substances. Under saturated conditions, these substances can precipitate due to changes in urine pH, temperature, metabolic disorders or lack of substances that inhibit crystal precipitation, forming urine crystals. The crystals that appear in urine mainly include calcium oxalate and calcium phosphate, as well as crystals precipitated from sulfonamides and other drugs. There are many types of proteins in urine, including albumin, transferrin, complement C3, α1-acid glycoprotein, α2 macroglobulin, IgG, IgA, IgM, etc., and albumin is only one of them. If the binding of albumin antigen-antibody is affected, the measurement results of urine microalbumin will be biased. Summary of the invention

[0004] In order to solve the above problems, the present invention provides an interference-resistant urine microalbumin assay kit, which is a liquid kit with strong stability, high accuracy, wide linear range and interference resistance.

[0005] The present invention is achieved through the following technical solutions:

[0006] An anti-interference urine microalbumin assay kit, the kit comprising reagent R1 and reagent R2;

[0007] Reagent R1 contains the following components:

[0008] Tris buffer 50-200mM

[0009] Sodium chloride 5g / L

[0010] EDTA-2Na 2g / L

[0011] Anti-interference additives 1-6g / L

[0012] Polyethylene glycol 8000 8g / L

[0013] Surfactant 1-4g / L

[0014] Pc300 1ml / L

[0015] Reagent R2 contains the following components:

[0016] Tris buffer 50-100mM

[0017] ALB antiserum 10-200mg / L

[0018] Dispersant 1-10 g / L

[0019] Stabilizer 1-10 g / L

[0020] Pc300 1ml / L

[0021] Preferably, the pH of the reagent R1 is 6.0-8.0; the pH of the reagent R2 is 6.0-8.0;

[0022] Preferably, the surfactant in the reagent R1 is one or both of Kao A60 and Kao A90;

[0023] Preferably, the anti-interference additives of the reagent R1 are polyhexamethylene biguanide, polyvinyl pyrrolidone, and sodium tetraborate in a mass ratio of 1:1:1.

[0024] Preferably, the stabilizer in the reagent R2 is an alkyl glycoside.

[0025] Preferably, the dispersant in the reagent R1 is sodium polyacrylate.

[0026] The preparation method of the kit of the present invention is:

[0027] (1) Preparation of reagent R1: Measure a volume of purified water equal to the preparation volume, calculate and weigh an appropriate amount of tris(hydroxymethyl)aminomethane buffer according to the preparation volume, adjust the pH, calculate and weigh an appropriate amount of sodium chloride, polyethylene glycol 8000, EDTA-2Na, Kao A60, polyhexamethylene biguanide, polyvinyl pyrrolidone, sodium tetraborate, and Pc300 according to the preparation volume, and stir well to obtain reagent R1;

[0028] (2) Preparation of reagent R2: Measure a volume of purified water equal to the preparation volume, calculate and weigh an appropriate amount of tris(hydroxymethyl)aminomethane buffer according to the preparation volume, adjust the pH, calculate and weigh an appropriate amount of ALB antiserum, alkyl glycoside, sodium polyacrylate, and Pc300 according to the preparation volume, and stir well to obtain reagent R2.

[0029] The above urine microalbumin assay kit is used to measure the concentration of urine microalbumin for non-disease diagnosis and treatment purposes.

[0030] Beneficial effects of the present invention:

[0031] 1. The surfactants in the reagent R1 of the present invention are Kao A90 and A60, which can effectively remove the chylomicron interference in urine, make the reagent R1 system uniformly dispersed, and greatly enhance the anti-interference ability of the reagent.

[0032] 2. The reagent R1 of the present invention adds anti-interference additives such as polyhexamethylene biguanide and polyvinyl pyrrolidone, which have obvious solubilizing and antibacterial effects, can eliminate turbidity caused by crystallization and turbidity, and adds sodium tetraborate to expose the antigen binding site, enhance the reaction specificity, and eliminate the interference of other proteins.

[0033] 3. The addition of stabilizer alkyl glycoside and dispersant sodium polyacrylate to the reagent R2 of the present invention not only improves the stability and dispersibility of the antiserum in the reagent R2 and avoids coagulation, but also makes the reagent R2 have excellent stability.

[0034] 4. The urine microalbumin determination reagent prepared by the present invention has good stability, strong anti-interference ability, high accuracy, and is significantly superior to similar products on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the correlation curve of the reagents of Example 1 and Comparative Example 1;

[0036] Figure 2 It is the correlation curve of the reagents of Comparative Example 1 and Comparative Example 5;

[0037] Figure 3 The concentration changes of the urine microalbumin determination reagents provided in Example 1 and Comparative Examples 1, 6, 7, 8, and 9 were subjected to stability tests. DETAILED DESCRIPTION

[0038] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0039] The kit of the present invention is used to determine the microalbuminuria in urine, and the test conditions are:

[0040] Method: endpoint method; main / sub wavelength: 340nm / 700nm; temperature: 37℃; correction type: nonlinear; calibration method: multi-point calibration; reaction direction: upward.

[0041] The specific operations are shown in Table 1.

[0042] Table 1 Urine microalbumin assay reagent operation steps

[0043]

[0044] Calculation results:

[0045]

[0046] Sample requirements:

[0047] 1. Fresh urine specimen or 24-hour urine

[0048] 2. Sample stability: Urine samples should be stored at 2-8°C before testing. If the sample cannot be tested immediately, it can be stored at 2-8°C for 2 days.

[0049] The present invention will be further described below in conjunction with specific embodiments:

[0050] Example 1

[0051] An anti-interference urine microalbumin assay kit, the kit comprising reagent R1 and reagent R2;

[0052] Reagent R1 contains the following components:

[0053] Tris buffer 50mmol / L

[0054] Sodium chloride 5g / L

[0055] EDTA-2Na 2g / L

[0056] Polyhexamethylene biguanide 2g / L

[0057] Polyvinylpyrrolidone 2g / L

[0058] Sodium tetraborate 2g / L

[0059] Polyethylene glycol 8000 8g / L

[0060] Kao A60 1g / L

[0061] Pc300 1ml / L

[0062] pH 7.0

[0063] Reagent R2 contains the following components:

[0064] Tris buffer 50mmol / L

[0065] ALB antiserum 100mg / L

[0066] Sodium polyacrylate 3g / L

[0067] Alkyl glycoside 3g / L

[0068] Pc300 1ml / L

[0069] The pH of reagent R2 is 7.0;

[0070] The preparation method of the above-mentioned urine microalbumin assay kit comprises the following steps:

[0071] a) Preparation of reagent R1: measure a volume of purified water equal to the preparation volume, calculate and weigh an appropriate amount of tris(hydroxymethyl)aminomethane buffer according to the preparation volume, adjust the pH to 7.0, calculate and weigh an appropriate amount of sodium chloride, polyethylene glycol 8000, Kao A60, polyhexamethylene biguanide, polyvinyl pyrrolidone, sodium tetraborate and other substances according to the preparation volume, and stir well to obtain reagent R1;

[0072] b) Preparation of reagent R2: Measure a volume of purified water equal to the preparation volume, calculate and weigh an appropriate amount of Tris buffer according to the preparation volume, adjust the pH to 7.0, calculate and weigh an appropriate amount of ALB antiserum, alkyl glycoside, sodium polyacrylate, and Pc300 according to the preparation volume, and stir well to obtain reagent R2.

[0073] Example 2

[0074] An anti-interference urine microalbumin assay kit, the kit comprising reagent R1 and reagent R2;

[0075] Reagent R1 contains the following components:

[0076] Tris buffer 50mmol / L

[0077] Sodium chloride 5g / L

[0078] EDTA-2Na 2g / L

[0079] Polyhexamethylene biguanide 2g / L

[0080] Polyvinylpyrrolidone 2g / L

[0081] Sodium tetraborate 2g / L

[0082] Polyethylene glycol 8000 8g / L

[0083] Kao A60 1g / L

[0084] Pc300 1ml / L

[0085] pH 7.0

[0086] Reagent R2 contains the following components:

[0087] Tris buffer 50mmol / L

[0088] ALB antiserum 100mg / L

[0089] Sodium polyacrylate 5g / L

[0090] Alkyl glycoside 5g / L

[0091] Pc300 1ml / L

[0092] The pH of reagent R2 is 7.0;

[0093] The preparation method of the above-mentioned urine microalbumin assay kit is the same as that of Example 1.

[0094] Example 3

[0095] An anti-interference urine microalbumin assay kit, the kit comprising reagent R1 and reagent R2;

[0096] Reagent R1 contains the following components:

[0097] Tris buffer 50mmol / L

[0098] Sodium chloride 5g / L

[0099] EDTA-2Na 2g / L

[0100] Polyhexamethylene biguanide 0.5g / L

[0101] Polyvinylpyrrolidone 0.5g / L

[0102] Sodium tetraborate 0.5g / L

[0103] Polyethylene glycol 8000 8g / L

[0104] Kao A60 2g / L

[0105] Pc300 1ml / L

[0106] pH 7.0

[0107] Reagent R2 contains the following components:

[0108] Tris buffer 50mmol / L

[0109] ALB antiserum 100mg / L

[0110] Sodium polyacrylate 5g / L

[0111] Alkyl glycoside 5g / L

[0112] Pc300 1ml / L

[0113] The pH of reagent R2 is 7.0;

[0114] The preparation method of the above-mentioned urine microalbumin assay kit is the same as that of Example 1.

[0115] Comparative Example 1

[0116] Imported Olympus urine microalbumin assay kit.

[0117] Comparative Example 2

[0118] The only difference from the urine microalbumin assay kit in Example 1 is that reagent R1 does not contain Kao A60, and the rest is the same as Example 1.

[0119] Comparative Example 3

[0120] The only difference from the urine microalbumin assay kit in Example 1 is that reagent R1 does not contain polyhexamethylene biguanide, and the rest is the same as Example 1.

[0121] Comparative Example 4

[0122] The only difference from the urine microalbumin assay kit in Example 1 is that reagent R1 does not contain polyvinyl pyrrolidone, and the rest is the same as Example 1.

[0123] Comparative Example 5

[0124] The only difference from the urine microalbumin assay kit in Example 1 is that reagent R1 does not contain sodium tetraborate, and the rest is the same as Example 1.

[0125] Comparative Example 6

[0126] The only difference from the urine microalbumin assay kit in Example 1 is that reagent R2 does not contain sodium polyacrylate, and the rest is the same as Example 1.

[0127] Comparative Example 7

[0128] The only difference from the urine microalbumin assay kit in Example 1 is that reagent R2 does not contain alkyl glycoside, and the rest is the same as Example 1.

[0129] Comparative Example 8

[0130] The only difference from the urine microalbumin assay kit in Example 1 is that reagent R2 does not contain sodium polyacrylate but contains an equal amount of sodium alginate, and the rest is the same as in Example 1.

[0131] Comparative Example 9

[0132] The only difference from the urine microalbumin assay kit in Example 1 is that reagent R2 does not contain alkyl glycoside but contains an equal amount of dextran, and the rest is the same as in Example 1.

[0133] Performance Verification

[0134] Experiment 1: Correlation Experiment

[0135] A correlation test was conducted, and the test scheme was as follows: Example 1, Comparative Example 1, and Comparative Example 5. 40 clinical urine samples were tested at the same time, and the correlation coefficient r was calculated by performing correlation analysis on the two groups of test results; the test results of Comparative Example 1 were used as the target value, and the relative deviations (Bias%) of the 40 pairs of data were calculated respectively. It was required that r was not less than 0.990, and the relative deviation was not more than ±10%.

[0136] Table 2 Correlation comparison experimental results

[0137]

[0138]

[0139] Table 3 Correlation coefficients of Comparative Example 1, Example 1 and Comparative Example 5

[0140]

[0141] From Table 2, Table 3 and Figure 1 , Figure 2 It can be seen that the maximum deviation of the urine sample determined by the reagents of Example 1 and Comparative Example 1 is 2.92%, and the correlation coefficient of the two reagents is 0.9981. The detection results of Example 1 and Comparative Example 1 are very close, and the correlation coefficient of Comparative Example 1 and Comparative Example 5 is 0.7961, and the difference in the detection results is large. Therefore, the detection reagent of Example 1 provided by the present invention has high accuracy, and the addition of sodium tetraborate is beneficial to exposing the antigen binding sites, so that the antigen-antibody binding specificity is enhanced, thereby improving the accuracy of the reagent.

[0142] Test 2: Stability test

[0143] The stability test of the urine microalbumin determination reagent provided in Example 1 and Comparative Examples 1, 6, 7, 8, and 9 was carried out. The test scheme was as follows: the reagents provided in Example 1 and Comparative Examples 1, 6, 7, 8, and 9 were placed in a 37°C water bath together, and the quality control product with a target value of 65±2.5 mg / L was detected every day, and the changes in the quality control product determination were monitored.

[0144] Table 4 Reagent thermal stability verification results (quality control)

[0145]

[0146] From Table 4 and Figure 3It can be seen that, similar to Comparative Example 1, the reagent in Example 1 provided by the present invention has good stability within 12 days under 37°C water bath conditions; while the quality control values ​​of the reagents in Comparative Examples 6 and 7 are significantly reduced under 37°C water bath conditions for 12 days, and the stability of the kit in Example 1 is better than that of the kits in Comparative Examples 6 and 7; although the stability of Comparative Examples 8 and 9 is improved by adding sodium alginate and dextran, it is still poorer than that of Example 1, so the addition of stabilizer alkyl glycoside and dispersant sodium polyacrylate is beneficial to improve the stability of reagent R2.

[0147] Test 3: Anti-interference test

[0148] Different concentrations of chyle, calcium oxalate and calcium phosphate interference samples were prepared, and the reagents provided in Example 1 and Comparative Examples 1, 2, 3 and 4 were used to measure samples with different concentrations of interfering substances added. The measurement result of urine microalbumin in level 1 sample was used as the base value, and the deviation % of the measured value of urine microalbumin in the interference sample from the base value was calculated. The deviation % ≥ ± 10% was considered interference. The results are shown in Tables 5 and 6.

[0149] Table 5 Calcium oxalate anti-interference test results

[0150]

[0151] Table 6 Calcium phosphate anti-interference experimental determination results

[0152]

[0153] Table 7 Chylomicron interference test results

[0154]

[0155] The results show that Example 1 is similar to Comparative Example 1. When the chyle in the sample to be tested is 1000 mg / dL, calcium oxalate and calcium phosphate have no interference with the determination of urine microalbumin when the concentration is 12 mmol / L. In Comparative Example 2, when the concentration of chyle in the sample to be tested is 400 mg / dl, it will interfere with the determination of urine microalbumin; in Comparative Examples 3 and 4, when the concentration of calcium oxalate and calcium phosphate in the sample to be tested is 8 mmol / L, it will interfere with the determination of urine microalbumin. Therefore, adding Kao A60 plays an important role in eliminating the interference of chyle, and adding polyhexamethylene biguanide and polyvinyl pyrrolidone is beneficial to eliminating the interference of increased turbidity caused by crystallization.

[0156] Experimental Four Linear Experiment

[0157] A sample with a high value of urine microalbumin was taken as 200 mg / L, and gradient dilution was performed to prepare 6 samples of different concentrations, which were 0 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L, respectively. The reagents provided in Example 1 and Comparative Examples 1, 6, and 8 were used for detection. Each sample at each concentration level was measured 3 times, and the average values ​​were taken. The test results are shown in Table 8:

[0158] Table 8 Reagent linearity verification results

[0159]

[0160] It can be seen from Table 8 that, similar to Comparative Example 1, Example 1 of the present invention changes linearly with the dilution concentration, and the linear correlation coefficient reaches 0.9986, indicating that Example 1 has a good linear range. Comparative Example 6 does not add the dispersant sodium polyacrylate, and the linearity is poor. Comparative Example 8 adds sodium alginate, and the linearity is improved, but it still does not meet the requirements. Therefore, adding the dispersant sodium polyacrylate can improve the linear range of the reagent.

[0161] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0162] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

Claims

1. An anti-interference urine microalbumin assay kit, characterized in that: The kit contains reagent R1 and reagent R2; Reagent R1 contains the following components: Tris buffer 50-200mM Sodium chloride 5g / L EDTA-2Na 2g / L Anti-interference additives 1-6g / L Polyethylene glycol 8000 8g / L Surfactant 1-4g / L Pc300 1ml / L Reagent R2 contains the following components: Tris buffer 50-100mM ALB antiserum 10-200mg / L Dispersant 1-10 g / L Stabilizer 1-10 g / L Pc300 1ml / L The pH of the reagent R1 is 6.0-8.0; the pH of the reagent R2 is 6.0-8.0; The surfactant in the reagent R1 is one or both of Kao A60 and Kao A90; The anti-interference additives of the reagent R1 are polyhexamethylene biguanide, polyvinyl pyrrolidone, and sodium tetraborate, with a mass ratio of 1:1:1; The stabilizer in the reagent R2 is an alkyl glycoside; The dispersant in the reagent R2 is sodium polyacrylate.

2. A method for preparing the urine microalbumin assay kit according to claim 1, characterized in that: The following steps are involved: a) Preparation of reagent R1: measure a volume of purified water equal to the preparation volume, calculate and weigh an appropriate amount of tris(hydroxymethyl)aminomethane buffer according to the preparation volume, adjust the pH, calculate and weigh an appropriate amount of sodium chloride, polyethylene glycol 8000, EDTA-2Na, Kao A60, polyhexamethylene biguanide, polyvinyl pyrrolidone, sodium tetraborate, and Pc300 according to the preparation volume, and stir well to obtain reagent R1; b) Preparation of reagent R2: measure a volume of purified water equal to the preparation volume, calculate and weigh an appropriate amount of Tris buffer according to the preparation volume, adjust the pH, calculate and weigh an appropriate amount of ALB antiserum, alkyl glycoside, sodium polyacrylate, and Pc300 according to the preparation volume, and stir well to obtain reagent R2.

3. Use of the urine microalbumin assay kit according to claim 1 for measuring the concentration of urine microalbumin for non-disease diagnosis and treatment purposes.

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