A kit for analyzing and detecting microalbumin in urine

Abstract

The present invention provides a urine microalbumin analysis and detection kit, belonging to the field of biotechnology. The present invention specifically optimizes the dilution buffer in the urine microalbumin detection reaction system, wherein the dilution buffer is a sample dilution buffer or a standard dilution buffer. By adjusting the specific component distribution ratio of the dilution buffer, the analysis and detection effect of oxidized albumin in urine is improved, so that the accuracy and sensitivity of the kit are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to an analytical detection kit for trace albumin in urine. Background Art

[0002] The occurrence of diabetic nephropathy (DN) is irreversible, and microalbuminuria (MAU) is an early sensitive indicator of kidney damage. MAU refers to the urinary albumin excretion rate between 30-300 mg / 24h or 20-200 μg / min. The MAU stage is an important subclinical transition stage between the non-clinical stage of DN (high filtration stage and asymptomatic stage) and the clinical stage (clinical nephropathy stage and end-stage renal disease stage). Therefore, MAU is sometimes also called latent DN or subclinical DN or early kidney disease. The MAU stage provides an excellent window for early diagnosis and effective treatment with reversibility for the prevention and treatment of DN. Detection of urinary microalbumin (ALB) is very important for judging the MAU stage.

[0003] ALB is commonly detected by immunofluorescence turbidimetry to detect microalbuminuria in patients. As disclosed in the prior art, urine is collected in the morning, and then the collected urine is centrifuged. After the centrifugation, the supernatant is extracted and placed in a centrifuge tube, and then detected by immunofluorescence turbidimetry. [1] . Turbidimetricinhibition immuno assay is a method for the dynamic determination of antigen-antibody binding. Its basic principle is: when the antigen and antibody react in a special dilution system and the ratio is appropriate (generally the antibody is in excess), the soluble immune complex formed will precipitate from the liquid phase under the action of the aggregation promoter (polyethylene glycol, etc.) in the dilution system, forming particles, causing turbidity in the reaction solution. When the antibody concentration is fixed, the amount of immune complex formed increases with the increase of the amount of antigen in the test sample, and the turbidity of the reaction solution also increases accordingly. By measuring the turbidity of the reaction solution and comparing it with a series of standards, the content of the antigen in the test sample can be calculated. However, due to the limitations of its own method, its accuracy is poor.

[0004] In the prior art, a relatively good method for detecting microalbuminuria is to detect it through ELISA, so there are many ELISA detection kits for detecting microalbuminuria on the market. These kits need to measure the standard curve when performing the test, and then complete the measurement of the sample. However, due to the stability problem of the system in the sample, the R value of the standard curve of these kits is relatively unstable and the R value is low, which causes the instability of the test results. In addition, the detection sensitivity of the existing ELISA detection kit for microalbuminuria needs to be further improved.

[0005] [1] Pan Changyu, Jin Wensheng. Strengthening the screening of microalbuminuria to improve the prevention and treatment of diabetic nephropathy[J]. Chinese Journal of Internal Medicine, 2004, 43(3):2. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a urine microalbumin analysis and detection kit for detecting the microalbumin content in a urine sample with high accuracy and high sensitivity.

[0007] In one aspect, the present invention provides a kit for analyzing and detecting urinary microalbumin.

[0008] The kit comprises a dilution buffer, which comprises: 0.01-0.02M phosphate, 0.2%-0.4% m / V sodium fluoride, 0.1%-0.3% m / V potassium chloride, and 0.5%-0.8% m / V vitamin B12.

[0009] The dilution buffer is a sample dilution buffer or a standard dilution buffer.

[0010] Preferably, the concentration of the phosphate is 0.01M or 0.02M.

[0011] Preferably, the concentration of sodium fluoride is 0.2%-0.3% m / V or 0.3%-0.4% m / V; more preferably, it is 0.2% m / V, 0.3% m / V or 0.4% m / V.

[0012] Preferably, the concentration of potassium chloride is 0.1%-0.2% m / V or 0.2%-0.3% m / V; more preferably, it is 0.1% m / V, 0.2% m / V, or 0.3% m / V.

[0013] Preferably, the vitamin B12 concentration is 0.5%-0.7% m / V, 0.5%-0.6% m / V, 0.6%-0.8% m / V, 0.6%-0.7% m / V, 0.7%-0.8% m / V; further preferably, it is 0.5% m / V, 0.7% m / V, 0.7% m / V, 0.7% m / V, 0.7% m / V.

[0014] Preferably, the phosphate is sodium dihydrogen phosphate.

[0015] The dilution buffer is used for sample dilution or standard dilution.

[0016] The dilution buffer also includes other components used for sample dilution or standard dilution.

[0017] The sample dilution multiple is 5-10 times.

[0018] Preferably, the kit is an ELISA kit.

[0019] The kit also includes other reagents necessary for ELISA detection, such as enzyme labeling reagent, color developer, stop solution, etc.

[0020] The kit also includes a standard substance.

[0021] The kit can be composed of an existing urine microalbumin ELISA kit and the aforementioned dilution buffer.

[0022] In another aspect, the present invention provides a dilution buffer.

[0023] The dilution buffer comprises: 0.01-0.02M phosphate, 0.2%-0.4% m / V sodium fluoride, 0.1%-0.3% m / V potassium chloride, and 0.5%-0.8% m / V vitamin B12.

[0024] Preferably, the concentration of the phosphate is 0.01M or 0.02M.

[0025] Preferably, the concentration of sodium fluoride is 0.2%-0.3% m / V or 0.3%-0.4% m / V; more preferably, it is 0.2% m / V, 0.3% m / V or 0.4% m / V.

[0026] Preferably, the concentration of potassium chloride is 0.1%-0.2% m / V or 0.2%-0.3% m / V; more preferably, it is 0.1% m / V, 0.2% m / V, or 0.3% m / V.

[0027] Preferably, the vitamin B12 concentration is 0.5%-0.7% m / V, 0.5%-0.6% m / V, 0.6%-0.8% m / V, 0.6%-0.7% m / V, 0.7%-0.8% m / V; further preferably, it is 0.5% m / V, 0.7% m / V, 0.7% m / V, 0.7% m / V, 0.7% m / V.

[0028] Preferably, the phosphate is sodium dihydrogen phosphate.

[0029] The dilution buffer is used for ELISA detection of urine samples.

[0030] Preferably, the diluent is used for ELISA detection of trace albumin in urine samples.

[0031] The dilution buffer is a sample dilution buffer or a standard dilution buffer.

[0032] In yet another aspect, the present invention provides use of the aforementioned dilution buffer in the preparation of a urine microalbumin detection kit.

[0033] Preferably, the kit is an ELISA detection kit.

[0034] The dilution buffer is used for sample dilution or standard dilution.

[0035] The sample dilution multiple is 5-10 times.

[0036] Preferably, the kit is an ELISA kit.

[0037] The kit also includes other reagents necessary for ELISA detection, such as enzyme labeling reagent, color developer, stop solution, etc.

[0038] The kit also includes the aforementioned standard substance.

[0039] Beneficial effects of the present invention:

[0040] The present invention optimizes the dilution buffer in the ELISA kit, so that the detection effect of the kit on urine samples is improved, the standard curve of the kit is more accurate, and the sample detection sensitivity is higher. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0042] Example 1 A kit for analyzing and detecting microalbumin in urine

[0043] This embodiment makes technical improvements to the existing finished kit, specifically an ELISA kit for human urine microalbumin (ALB) of ELISA, with the item number ml025063.

[0044] The detection steps of the ELISA kit before improvement are as follows:

[0045] 1. Collect the urine sample in a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate is formed during storage, centrifuge it again.

[0046] 2. Adding standard samples: Set up standard sample wells and sample wells, and add 50 μL of standard samples of different concentrations to each standard sample well (diluted with standard sample diluent).

[0047] 3. Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40μL of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10μL of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0048] 4. Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0049] 5. Liquid preparation: Dilute the 30 times concentrated washing solution with 30 times distilled water and set aside.

[0050] 6. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0051] 7. Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well.

[0052] 8. Incubation: Same operation as 4.

[0053] 9. Washing: Same operation as 6.

[0054] 10. Color development: First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake to mix, and develop the color at 37℃ in the dark for 15 minutes.

[0055] 11. Termination: Add 50 μL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0056] 12. Determination: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0057] 13. Draw a standard curve on the coordinate paper with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. Find the corresponding concentration from the standard curve according to the OD value of the sample; then multiply it by the dilution factor; or use the concentration and OD value of the standard to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of the sample.

[0058] The R value of the correlation coefficient between the linear regression of the standard curve disclosed by the kit and the expected concentration is above 0.95. In actual testing, the R value of this batch of products is 0.9607.

[0059] In the improved kit, the following concentrations of drugs are added to the sample dilution in step 3: 0.02M sodium dihydrogen phosphate, 0.3% m / V sodium fluoride, 0.2% m / V potassium chloride, 0.6% m / V vitamin B12. At the same time, each drug is dissolved in the standard diluent at the above concentration, that is, the following final concentrations of drugs are added to the standard diluent: 0.02M sodium dihydrogen phosphate, 0.3% m / V sodium fluoride, 0.2% m / V potassium chloride, 0.6% m / V vitamin B12.

[0060] The R value of the correlation coefficient between the linear regression of the improved test kit samples and the expected concentration is 0.9882, which is significantly higher than the improvement, indicating that the dilution buffer of this embodiment improves the detection accuracy of the test kit.

[0061] Example 2 A kit for analyzing and detecting microalbumin in urine

[0062] The difference with respect to Example 1 is that the standard buffer contains the following components: 0.01 M sodium dihydrogen phosphate, 0.3% m / V sodium fluoride, 0.2% m / V potassium chloride, and 0.7% m / V vitamin B12.

[0063] Referring to the detection method of Example 1, the correlation coefficient R value between the linear regression of the kit samples and the expected concentration was 0.9837.

[0064] Example 3 A urine sample ELISA detection dilution buffer

[0065] The difference with respect to Example 1 is that the standard buffer contains the following components: 0.02M sodium dihydrogen phosphate, 0.2% m / V sodium fluoride, 0.3% m / V potassium chloride, and 0.5% m / V vitamin B12.

[0066] Referring to the detection method of Example 1, the correlation coefficient R value between the linear regression of the kit samples and the expected concentration was 0.9841.

[0067] Example 4 Kit sensitivity.

[0068] The standard in Example 1 was used as a sample, first diluted with sterile water to different concentrations (5ng / L, 1ng / L, 0.5ng / L, 0.1ng / L, 0.05ng / L), and then tested using the kits before and after improvement. The results showed that the kit before improvement could detect a minimum of 0.5ng / L of standard, and after improvement, it could detect a minimum of 0.1ng / L of sample concentration, with high sensitivity.

[0069] Example 5 Sample Test

[0070] The two kits in Example 1 (before improvement: the sample diluent and the standard diluent were prepared by the original kit; after improvement: the sample diluent and the standard diluent were added with improved components) were used to test urine samples from 20 healthy volunteers. The final test results are as follows:

[0071]

[0072] The above results show that the kit of the present application is feasible for use in reagent detection.

[0073] Comparative Example

[0074] A comparative example is set with reference to Example 1, as follows:

[0075]

[0076]

[0077] The results of the linear regression and the expected concentration correlation coefficient R value of the samples in comparative examples 1-4 are as follows:

[0078] Comparative Example R-Value 1 0.9587 2 0.9594 3 0.9613 4 0.9591

[0079] Comparative Examples 5-8 were tested for sensitivity with reference to Example 5, and the results were as follows:

[0080] Comparative Example Sensitivity 5 0.5ng / L 6 1ng / L 7 0.5ng / L 8 0.5ng / L

[0081] When the combination of the four reagents of the present invention is applied to sample detection, the detection sensitivity of the test kit can be improved. When applied to standard products, the accuracy of the standard curve of the test kit can be improved. It is speculated that the binding ability of the antibody antigen in the ELISA test kit is affected, so for the sample, a lower concentration can be detected, and for the standard product, it is made to combine more fully, so that the measured data is more accurate, and the accuracy of the standard curve is higher. Sodium dihydrogen phosphate is often used to prepare phosphate buffer, sodium fluoride and potassium chloride are common salts, together with vitamin B12, the above four substances do not show the above effects when used alone, and combined application and under a specific concentration combination, there is an unexpected effect.

[0082] Those skilled in the art know that the embodiments disclosed in this application are only examples of the technical solutions covered by the present invention and are not intended to limit the present invention. Those skilled in the art who replace conventional technical means on the basis of the above embodiments to achieve the technical effects of the present application are also within the technical scope of protection of this application. Although only one kit was tested in the embodiments of the present invention, according to the above analysis, those skilled in the art can apply it to other detection kits of the same type and expect its technical effects, so the scope of application of the present invention is not limited to the kit tested in the embodiments.

Claims

1. A kit for analyzing and detecting microalbuminuria, characterized in that: The kit includes a dilution buffer, which is composed of the following components: 0.01-0.02M phosphate, 0.2%-0.4% m / V sodium fluoride, 0.1%-0.3% m / V potassium chloride and 0.5%-0.8% m / V vitamin B12; the dilution buffer is a sample dilution buffer and a standard dilution buffer; the kit also includes other ELISA detection reagents.

2. The kit according to claim 1, characterized in that The ELISA detection reagent is one or more of an enzyme-labeled reagent, a color developer, and a stop solution.

3. The kit according to claim 2, characterized in that The kit also includes a standard substance.

4. Use of a dilution buffer in the preparation of an ELISA kit for the analysis and detection of urine microalbumin; the dilution buffer comprises: 0.01-0.02M phosphate, 0.2%-0.4% m / V sodium fluoride, 0.1%-0.3% m / V potassium chloride and 0.5%-0.8% m / V vitamin B12; the dilution buffer is a sample dilution buffer and a standard dilution buffer; the dilution buffer is used for ELISA detection of urine samples.

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