CN115754301A - Urine microalbumin analysis and detection kit - Google Patents
Urine microalbumin analysis and detection kit Download PDFInfo
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- CN115754301A CN115754301A CN202211434440.9A CN202211434440A CN115754301A CN 115754301 A CN115754301 A CN 115754301A CN 202211434440 A CN202211434440 A CN 202211434440A CN 115754301 A CN115754301 A CN 115754301A
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Abstract
The invention provides an analytical detection kit for microalbuminuria, belonging to the technical field of biology. The invention specifically optimizes a dilution buffer solution in a urine microalbumin detection reaction system, wherein the dilution buffer solution is a sample dilution buffer solution or a standard substance dilution buffer solution. By adjusting the specific component ratio of the dilution buffer, the analysis and detection effect of the oxidized albumin in urine is improved, and the accuracy and sensitivity of the kit are improved.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an analysis and detection kit for microalbumin in urine.
Background
The occurrence of Diabetic Nephropathy (DN) is irreversible, while Microalbuminuria (MAU) is an early sensitive indicator of kidney injury, and MAU means that the urinary albumin excretion rate is between 30-300mg/24h or 20-200 mug/min. The MAU stage is an important subclinical transition stage between the nonclinical stage (hypercatemic and asymptomatic) and clinical stages (clinical and terminal nephrotic stages) of DN, and thus MAU is sometimes also referred to as recessive DN or subclinical DN or early renal disease. The MAU phase provides an excellent window of early diagnosis and effective treatment for DN with reversibility. Detection of urinary microalbumin (ALB) is important for determining MAU stage.
A commonly used assay for ALB is the detection of microalbumin in the urine of a patient by immunoturbidimetry, as disclosed in the prior art: collecting urine of a patient in the morning, centrifuging the collected urine, extracting supernatant after centrifugation, placing the supernatant into a centrifuge tube, and detecting by an immunoturbidimetric assay [1] . Immunoturbidimetry (Turbidimetric inhibition immunoassay) is a method for measuring the binding kinetics of an antigen and an antibody. The basic principle is as follows: when the antigen and antibody react in a special dilution system and the ratio is appropriate (generally, it is specified that the antibody is in excess), the formed soluble immune complex precipitates from the liquid phase under the action of a polymerization promoter (polyethylene glycol or the like) in the dilution system to form microparticles, and turbidity appears in the reaction solution. When the antibody concentration is fixed, the amount of the immunocomplex formed increases with the increase in the amount of the antigen in the sample, and the turbidity of the reaction solution also increases. The content of the antigen in the sample can be calculated by measuring the turbidity of the reaction solution and comparing with a series of standard products. But the accuracy is poor due to the limitation of the method.
In the prior art, a method for detecting urine microalbumin with a good detection effect is to detect the urine microalbumin by ELISA, so a plurality of ELISA detection kits for detecting the urine microalbumin exist in the market. The detection of the kits requires the measurement of a standard curve to complete the measurement of a sample, but the R value of the standard curve is unstable and is low due to the problem of system stability in the sample, which causes instability of the detection result. In addition, the detection sensitivity of the existing ELISA detection kit for detecting urine trace urine protein needs to be further improved.
[1] Pan Changyu, jin wen sheng, screening for enhanced microalbuminuria improved the level of prevention and treatment of diabetic nephropathy [ J ]. J.Zhonghua J.J.J.Chi.J. 2004,43 (3): 2.
Disclosure of Invention
In order to solve the problems, the invention provides an analysis and detection kit for urine microalbumin, which is used for detecting the microalbumin content in a urine sample with high precision and high sensitivity.
In one aspect, the invention provides a kit for analyzing and detecting urinary microalbumin.
The kit comprises a dilution buffer solution, wherein the dilution buffer solution comprises: 0.01-0.02M phosphate, 0.2-0.4% M/V sodium fluoride, 0.1-0.3% M/V potassium chloride, 0.5-0.8% M/V vitamin B12.
The dilution buffer is a sample dilution buffer or a standard dilution buffer.
Preferably, the phosphate is present at a concentration of 0.01M or 0.02M.
Preferably, the concentration of sodium fluoride is 0.2% -0.3% m/V or 0.3% -0.4% m/V; more preferably 0.2% m/V, 0.3% m/V or 0.4% m/V.
Preferably, said potassium chloride is present in a concentration of 0.1% -0.2% m/V or 0.2% -0.3% m/V; more preferably 0.1% m/V, 0.2% m/V, 0.3% m/V.
Preferably, said 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; more preferably 0.5% m/V, 0.7% m/V.
Preferably, the phosphate is sodium dihydrogen phosphate.
The dilution buffer is used for sample dilution or standard dilution.
The dilution buffer also comprises other components for sample dilution or standard dilution.
The dilution factor of the sample is 5-10 times.
Preferably, the kit is an ELISA kit.
The kit also comprises other reagents necessary for ELISA detection: such as enzyme labeling reagent, color developing agent, stop solution, etc.
The kit also comprises a standard substance.
The kit can be composed of the prior urine microalbumin ELISA kit and the dilution buffer solution.
In another aspect, the invention provides a dilution buffer.
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, 0.5-0.8% M/V vitamin B12.
Preferably, the phosphate is present at a concentration of 0.01M or 0.02M.
Preferably, the concentration of sodium fluoride is 0.2% -0.3% m/V or 0.3% -0.4% m/V; more preferably 0.2% m/V, 0.3% m/V or 0.4% m/V.
Preferably, the concentration of potassium chloride is 0.1% -0.2% m/V or 0.2% -0.3% m/V; more preferably 0.1% m/V, 0.2% m/V, 0.3% m/V.
Preferably, said 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; more preferably 0.5% m/V, 0.7% m/V.
Preferably, the phosphate is sodium dihydrogen phosphate.
The dilution buffer is used for ELISA detection of urine samples.
Preferably, the diluent is used for ELISA detection of microalbumin in urine samples.
The dilution buffer is a sample dilution buffer or a standard dilution buffer.
In still another aspect, the invention provides the use of the aforementioned dilution buffer in the preparation of a urine microalbumin assay kit.
Preferably, the kit is an ELISA detection kit.
The dilution buffer is used for sample dilution or standard dilution.
The dilution factor of the sample is 5-10 times.
Preferably, the kit is an ELISA kit.
The kit also comprises other reagents necessary for ELISA detection: such as enzyme labeling reagent, color developing agent, stop solution, etc.
The kit also comprises the standard substance.
The invention has the beneficial effects that:
according to the invention, by optimizing the dilution buffer solution in the ELISA kit, the detection effect of the kit on the urine sample is improved, the standard curve of the kit is more accurate, and the sample detection sensitivity is higher.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples, unless otherwise specified, and experimental methods not specified in specific conditions in the examples, are generally commercially available according to conventional conditions, and materials, reagents, and the like used in the following examples, unless otherwise specified.
Example 1 analysis and detection kit for microalbuminuria
The present embodiment is a technical improvement of the existing finished product kit, and specifically relates to an enzyme-linked biological human urine microalbumin (ALB) ELISA kit with a product number of ml025063.
The detection steps of the ELISA kit before improvement are as follows:
1. urine samples were collected using a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). The supernatant was carefully collected and stored for another centrifugation if a precipitate formed.
2. Sample adding of the standard: standard wells and sample wells were set, and 50 μ L of standard (diluted with standard diluent) was added to each standard well at different concentrations.
3. Sample adding: blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way) and sample holes to be detected are respectively arranged. And (3) adding 40 mu L of sample diluent into the sample hole to be detected on the enzyme-labeled coated plate, and then adding 10 mu L of sample to be detected (the final dilution of the sample is 5 times). Adding sample to the bottom of the plate hole of the enzyme label, keeping the sample from touching the hole wall as much as possible, and gently shaking and mixing the sample and the hole wall.
4. And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 minutes.
5. Preparing liquid: and diluting the 30 times of concentrated washing liquid by 30 times of distilled water for later use.
6. Washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry.
7. Adding an enzyme: 50 μ L of enzyme-labeled reagent was added to each well, except for blank wells.
8. And (3) incubation: the operation is the same as 4.
9. Washing: the operation is the same as 6.
10. Color development: 50 mu L of color developing agent A is added into each hole, 50 mu L of color developing agent B is added into each hole, the mixture is evenly mixed by gentle shaking, and the mixture is developed for 15 minutes in a dark place at 37 ℃.
11. And (4) terminating: the reaction was stopped by adding 50. Mu.L of stop solution to each well (blue color turned to yellow color).
12. And (3) determination: the absorbance (OD value) of each well was measured sequentially at a wavelength of Kong Diaoling, 450nm, as a blank. The measurement should be performed within 15 minutes after the addition of the stop solution.
13. Drawing a standard curve on coordinate paper by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate, and finding out the corresponding concentration from the standard curve according to the OD value of the sample; multiplying by the dilution times; or calculating a linear regression equation of the standard curve by using the concentration and OD value of the standard substance, substituting the OD value of the sample into the equation to calculate the concentration of the sample, and multiplying the concentration by the dilution factor to obtain the actual concentration of the sample.
The standard curve linear regression disclosed by the kit has a correlation coefficient R value with the expected concentration of more than 0.95, and in actual detection, the R value of the product of the batch is 0.9607.
In the improved kit, the following medicines are added in the sample dilution in the 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. Simultaneously dissolving each drug in the standard dilution at the above concentration, i.e. adding the following drugs at the final concentration to the standard dilution: 0.02M sodium dihydrogen phosphate, 0.3% M/V sodium fluoride, 0.2% M/V potassium chloride, 0.6% M/V vitamin B12.
The improved kit sample linear regression and expected concentration correlation coefficient R value is 0.9882, which is obviously improved compared with the improvement, and the dilution buffer solution of the embodiment improves the detection accuracy of the kit.
Example 2 analysis and detection kit for microalbuminuria
The difference with respect to example 1 is that the standard buffer contains the following components: 0.01M sodium dihydrogen phosphate, 0.3% M/V sodium fluoride, 0.2% M/V potassium chloride, 0.7% M/V vitamin B12.
Referring to the assay method of example 1, the coefficient of linear regression of the kit sample to the expected concentration, R, was 0.9837.
Example 3 dilution buffer for ELISA detection of urine sample
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, 0.5% M/V vitamin B12.
Referring to the assay method of example 1, the coefficient of linear regression of the kit sample to the expected concentration, R, was 0.9841.
Example 4 kit sensitivity.
The standard substance in the example 1 is used as a sample, the sample is firstly diluted to different concentrations (5 ng/L, 1ng/L, 0.5ng/L, 0.1ng/L and 0.05 ng/L) by sterile water, and then the kit before and after the improvement is used for detection, and the result shows that the kit can detect the standard substance with the lowest concentration of 0.5ng/L before the improvement of the batch, can detect the sample with the lowest concentration of 0.1ng/L after the improvement, and has higher sensitivity.
Example 5 sample testing
Urine samples from 20 healthy volunteers were tested using the two kits of example 1 (before modification: sample diluent and standard diluent were both self-contained in the original kit; after modification: modified components were added to the sample diluent and the standard diluent), and the final test results were as follows:
the above results indicate that the kit of the present application is feasible for application in reagent detection.
Comparative example
A comparative example was set up with reference to example 1, as follows:
the results of the measurement of the correlation coefficient R values between the linear regression and the expected concentrations of the samples of the comparative examples 1 to 4 are as follows:
| comparative example | R value |
| 1 | 0.9587 |
| 2 | 0.9594 |
| 3 | 0.9613 |
| 4 | 0.9591 |
Comparative examples 5 to 8 the detection sensitivity was as follows with reference to example 5:
| comparative example | Sensitivity of the probe |
| 5 | 0.5ng/L |
| 6 | 1ng/L |
| 7 | 0.5ng/L |
| 8 | 0.5ng/L |
When the combination of the four reagents is applied to sample detection, the detection sensitivity of the kit can be improved. When the kit is applied to a standard substance, the accuracy of a standard curve of the kit can be improved, and the binding capacity of the antibody antigen in the ELISA kit is influenced, so that a sample can be detected at a lower concentration, and the standard substance is bound more sufficiently, so that the measured data is more accurate, and the accuracy of the standard curve is higher. Sodium dihydrogen phosphate is commonly used for preparing a phosphate buffer solution, sodium fluoride and potassium chloride are common salts and are used together with vitamin B12, the four substances do not show the effect when being used independently, and the two substances have unexpected effect when being combined and combined under a specific concentration.
Those skilled in the art will appreciate that the embodiments disclosed in the present 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 can substitute conventional technical means based on the above embodiments to achieve the technical effects of the present application, and the technical scope of the present application is also within the technical scope of the present application. Although only one kind of kit is tested in the examples of the present invention, those skilled in the art can apply the test kit to other similar types of test kits according to the above analysis and expect the technical effects thereof, so that the scope of application of the present invention is not limited to the kit tested in the examples.
Claims (10)
1. The kit for analyzing and detecting the microalbumin in urine is characterized by comprising a dilution buffer solution, wherein the dilution buffer solution 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.
2. The kit according to claim 1, wherein the dilution buffer is a sample dilution buffer or a standard dilution buffer.
3. The kit of claim 1, wherein the kit is an ELISA detection kit.
4. The kit of claim 2, wherein the dilution buffer further comprises other components for sample dilution or standard dilution.
5. The kit of claim 3, wherein the kit further comprises other ELISA detection reagents.
6. The kit of claim 5, wherein the ELISA detection reagent is one or more of an enzyme-labeled reagent, a color-developing agent and a stop solution.
7. The kit of claim 6, further comprising a standard.
8. A dilution buffer, comprising: 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.
9. The dilution buffer according to claim 10, which is a sample dilution buffer or a standard dilution buffer.
10. Use of the dilution buffer according to any one of claims 8 to 9 for the preparation of an analytical test kit for urinary microalbumin.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211434440.9A CN115754301A (en) | 2022-11-16 | 2022-11-16 | Urine microalbumin analysis and detection kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211434440.9A CN115754301A (en) | 2022-11-16 | 2022-11-16 | Urine microalbumin analysis and detection kit |
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| CN115754301A true CN115754301A (en) | 2023-03-07 |
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| CN202211434440.9A Pending CN115754301A (en) | 2022-11-16 | 2022-11-16 | Urine microalbumin analysis and detection kit |
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