CN108120714B - Reagent for detecting microalbumin in urine and application thereof - Google Patents
Reagent for detecting microalbumin in urine and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of medical detection, and discloses a reagent for detecting urine microalbumin and application thereof. The reagent comprises a solution A and a solution B, wherein the solution A is a sodium oxalate buffer solution, and the sodium oxalate buffer solution contains sodium oxalate and one or two selected from sodium molybdate and sucrose; and the solution B takes tetrahydrofuran and toluene as solvents and contains citric acid, polypropylene glycol and phenolphthalein dye. According to the invention, through adjusting and optimizing the components in the reagent, the influence of various interference factors in the urine sample on the detection is reduced, higher accuracy and precision are achieved, the linear relation is good in a large range, the reliability is high, and the defect that the conventional like product is insufficient in accuracy and precision is overcome.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a reagent for detecting microalbumin in urine and application thereof.
Background
Urine microalbumin is a microalbumin that occurs in urine, which is normally present in very small amounts. Urine microalbumin is the gold standard for early kidney injury, since it alters the ability of the kidney to filter albumin when the kidney becomes diseased, leading to leakage of albumin into the urine.
Currently, the detection methods of urine microalbumin are as follows: wet chemical quantitative detection, dry chemical qualitative detection, dry chemical semi-quantitative detection and the like, wherein the wet chemical quantitative detection consumes longer time according to different detection time of different full-automatic biochemical analyzers; dry chemical semi-quantitative detection is more suitable for coarse screening.
At present, test paper for detecting microalbumin on the market is known as Korea Saibao, but belongs to a semi-quantitative detection method, can only give an approximate content range, cannot give an accurate content, and has high false positive.
Chinese patent CN103901034A discloses a reagent strip for detecting microalbumin in urine, which can perform both semi-quantitative detection and quantitative detection, but improper reagent selection easily results in insufficient precision and accuracy of detection results.
Disclosure of Invention
In view of the above, the present invention is to provide a reagent for detecting urine microalbumin, which has high accuracy and precision when detecting urine microalbumin;
another object of the present invention is to provide a test strip based on the above reagent, which has high accuracy and precision when detecting microalbumin in urine, reduces false positive results, and simultaneously can perform quantitative and semi-quantitative detection;
another object of the present invention is to provide a quantitative or semi-quantitative assay method based on the above reagent, which has high accuracy and precision in assaying microalbumin in urine.
In order to achieve the above purpose, the invention provides the following technical scheme:
a reagent for detecting urine microalbumin comprises a solution A and a solution B, wherein the solution A is a sodium oxalate buffer solution, and the sodium oxalate buffer solution contains sodium oxalate and one or two selected from sodium molybdate and sucrose;
and the solution B takes tetrahydrofuran and toluene as solvents and contains citric acid, polypropylene glycol and sulfophthalein dye.
Aiming at the defects that the existing product for detecting the urine microalbumin is insufficient in the aspects of accuracy and precision, the invention obviously improves the precision and the accuracy in detection by adjusting the composition of the detection reagent.
Preferably, the pH value of the sodium oxalate buffer solution is 3.0-3.4. In a specific embodiment of the present invention, the pH value of the sodium oxalate buffer is adjusted by adding an amount of sodium oxalate, for example, the concentration of sodium oxalate is 133g/1000mL, and the pH value is 3.2.
Preferably, the concentration of sodium molybdate in the sodium oxalate buffer solution is 2.1-2.3% (w/v), and the concentration of sucrose is 1.2-1.3% (w/v). In a specific embodiment of the invention, the sodium molybdate is present at a concentration of 2.1% or 2.3% (w/v), and the sucrose is present at a concentration of 1.2% or 1.3% (w/v); more specifically, the sodium oxalate buffer solution contains sodium molybdate, and the concentration of the sodium molybdate is 2.1% (w/v); or the sodium oxalate buffer comprises sucrose with the concentration of 1.2% (w/v); or the sodium oxalate buffer solution contains sodium molybdate and sucrose, the concentration of the sodium molybdate is 2.3 percent (w/v), and the concentration of the sucrose is 1.3 percent (w/v); in the invention, other interfering components such as ions, urea and the like in urine are removed by adding sodium molybdate, and the reaction system can be diffused more uniformly during detection by adding sucrose, so that the detection result is more accurate and accurate.
Preferably, the solution B takes tetrahydrofuran and toluene as solvents, and comprises 1.0-1.4% (w/v) of citric acid, 3.0-5.0% (v/v) of polypropylene glycol and 0.04-0.10% (w/v) of sulfophthalein dye. According to the invention, on the basis of conventional tetrahydrofuran, methanol, ethanol and other solvents, toluene is adopted to replace methanol or ethanol, so that a more stable reaction environment is provided for a reaction system, and the influence on a detection result is reduced.
In a specific embodiment of the invention, the solution B takes tetrahydrofuran and toluene as solvents, and comprises 1.0% (w/v) of citric acid, 3.0% (v/v) of polypropylene glycol, and 0.04% (w/v) of sulfophthalein dye; or tetrahydrofuran and toluene as solvent, 1.2% (w/v) citric acid, 4.0% (v/v) polypropylene glycol, 0.06% (w/v) sulfophthalein dye; or tetrahydrofuran and toluene as solvent, 1.3% (w/v) citric acid, 5.0% (v/v) polypropylene glycol, 0.08% (w/v) sulfophthalein dye; preferably, the volume ratio of the hydrofuran to the toluene is 1: (1-1.5).
Meanwhile, the invention also provides application of the reagent in preparing a test strip for detecting urine microalbumin or application in detecting urine microalbumin.
The invention provides a test strip for detecting urine microalbumin, which is prepared by soaking carrier filter paper in solution A and solution B of the reagent uniformly in sequence and then drying. More specifically, the preparation process is as follows:
soaking the carrier filter paper in the solution A uniformly, absorbing the redundant solution, and drying in a drying oven at 100 ℃ for 30 min;
soaking the dried filter paper soaked with the solution A in the solution B uniformly, absorbing the redundant solution, and drying in a drying oven at 60 ℃ for 30 min.
For convenient operation and carrying, the test strip can be cut into strips, adhered to a PVC sheet and then cut into suitable strips, and the specification and the shape of a final product can be adjusted at will, which depends on actual requirements.
Preferably, the support filter paper is 3MM or 3-grade chromatography filter paper.
According to the prepared test strip, the invention can also carry out quantitative or semi-quantitative detection on the urine sample, wherein the semi-quantitative detection method comprises the following steps:
respectively dropwise adding bovine serum albumin solution with standard gradient concentration onto the test strip to obtain a standard colorimetric card;
and dripping the urine sample on the test strip, and comparing the corresponding relation between the color and the concentration on the standard colorimetric card according to the color development result to obtain a semi-quantitative result.
More specifically, a bovine serum albumin standard solution with the concentration of 320mg/L is prepared and is diluted into 7 concentrations of 320mg/L, 160mg/L, 80mg/L, 40mg/L, 20mg/L, 10mg/L and 0mg/L in a gradient way; adding the bovine serum albumin solution with the standard gradient concentration on a test strip, wherein the sample size is 30ul, and observing after 90 seconds, the obvious color gradation from colorless to dark blue can be observed by naked eyes to obtain a standard colorimetric card; and adding a sample on the test strip, wherein the sample amount is 30ul, comparing with a standard colorimetric card, and visually observing the concentration corresponding to the standard colorimetric card closest to the sample card, namely the concentration of the sample to be detected.
The method for quantitatively detecting the microalbumin in the urine comprises the following steps:
respectively dripping bovine serum albumin solution with standard gradient concentration on the test strip, and drawing a standard curve according to a urine analyzer;
and dripping a urine sample on the test strip, obtaining a detection value through a urine analyzer, and obtaining the corresponding microalbumin concentration according to a standard curve.
More specifically, the test strip is put into a special card shell and a card box and is placed in a reagent card chamber of a special full-automatic urine analyzer, the sample size is 30ul, and the reaction time is 90 seconds;
placing the bovine serum albumin solution with the standard gradient concentration in a sample tray of a full-automatic urine analyzer, detecting one by one, and drawing a standard curve;
and (3) placing the sample in a sample tray of the full-automatic urine analyzer, and after the sample is detected to be finished, automatically calculating the concentration of the detected sample by the analyzer according to the standard curve.
According to the actual detection test result, the method has higher accuracy, and the relative deviation is less than 5 percent and far lower than 10 percent of the standard requirement; the precision result shows that the standard deviation rate is 3.1 percent and is far less than 10 percent of the standard requirement, which indicates that the method has high precision; the results of the linear relationship test show that the linear relationship has good linearity in the range of 0.0 mg/L-320.0 mg/L, and the R value is 0.9999 and is close to 1.
In addition, the components in the similar products are replaced or the components in the similar products are deleted on the components of the reagent, and the comparison of the components of the comparison reagent products shows that the accuracy and the precision of the comparison products are greatly different, the correlation R value is 0.9347, and the CV value exceeds 10 percent of the requirement.
According to the technical scheme, the influence of various interference factors in the urine sample on detection is reduced by adjusting and optimizing components in the reagent, high accuracy and precision are achieved, the linear relation is good in a large range, the reliability is high, and the defects that the existing similar products are insufficient in accuracy and precision are overcome.
Drawings
FIG. 1 shows a standard color chart; wherein, the corresponding concentration is below the color block, and the unit is mg/L.
Detailed Description
The invention discloses a reagent for detecting urine microalbumin and application thereof, and a person skilled in the art can realize the detection by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the reagents and their associated applications of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications, or appropriate alterations and combinations of the reagents and their associated applications, described herein may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
The reagent for detecting urinary creatinine and the application thereof provided by the invention are further described below.
Example 1: the reagent and the test strip of the invention
1. Reagent composition
The solution A is: 133g of sodium oxalate, 21g of sodium molybdate (2.1 percent) and purified water are added to 1000mL, and the pH value is 3.2.
The solution B is: 10g of citric acid (1.0%), 30mL of polypropylene glycol (3.0%), 400mg of sulfophthalein dye (0.04%), and a volume of 1000mL of tetrahydrofuran and toluene (volume ratio 1: 1) was added.
2. Preparation of test paper strip
Soaking filter paper in the solution A uniformly, absorbing the redundant solution, and drying in a drying oven at 100 ℃ for 30 min;
soaking the dried filter paper soaked with the solution A in the solution B uniformly, absorbing the redundant solution, and drying in a drying oven at 60 ℃ for 30 min.
The dried filter paper is cut into strips, stuck on a PVC sheet and cut into proper strips.
Example 2: the reagent and the test strip of the invention
1. Reagent composition
The solution A is: 133g of sodium oxalate, 12g of sucrose (1.2%), purified water to 1000mL, and pH 3.2.
The solution B is: 12g citric acid (1.2%), 40mL polypropylene glycol (4.0%), 600mg sulfophthalein dye (0.06%), and a volume of 1000mL with tetrahydrofuran and toluene (1: 1.5 by volume).
2. Preparation of test paper strip
Soaking filter paper in the solution A uniformly, absorbing the redundant solution, and drying in a drying oven at 100 ℃ for 30 min;
soaking the dried filter paper soaked with the solution A in the solution B uniformly, absorbing the redundant solution, and drying in a drying oven at 60 ℃ for 30 min.
The dried filter paper is cut into strips, stuck on a PVC sheet and cut into proper strips.
Example 3: the reagent and the test strip of the invention
1. Reagent composition
The solution A is: 133g of sodium oxalate, 23g of sodium molybdate (2.3%), 13g of sucrose (1.3%), and purified water to 1000mL with a pH value of 3.2.
The solution B is: 13g citric acid (1.3%), 50mL polypropylene glycol (5.0%), 800mg sulfophthalein dye (0.08%), and a volume of 1000mL with tetrahydrofuran and toluene (1: 1.5 by volume).
2. Preparation of test paper strip
Soaking filter paper in the solution A uniformly, absorbing the redundant solution, and drying in a drying oven at 100 ℃ for 30 min;
soaking the dried filter paper soaked with the solution A in the solution B uniformly, absorbing the redundant solution, and drying in a drying oven at 60 ℃ for 30 min.
The dried filter paper is cut into strips, stuck on a PVC sheet and cut into proper strips.
Example 4: semi-quantitative detection method
(1) Bovine serum albumin standard solution with the concentration of 320mg/L is prepared and is diluted into 7 concentrations of 320mg/L, 160mg/L, 80mg/L, 40mg/L, 20mg/L, 10mg/L and 0mg/L in a gradient way.
(2) The standard solution is added to a test strip, the sample size is 30ul, and after 90 seconds, a clear color gradation from colorless to dark blue can be observed by naked eyes.
(3) And adding a sample on the test strip, wherein the sample amount is 30ul, comparing with a standard colorimetric card, and visually observing the concentration corresponding to the standard colorimetric card closest to the sample card, namely the concentration of the sample to be detected. The standard color chart is shown in figure 1.
Example 5: quantitative detection method
(1) The test strip is put into a special card shell and a card box and is placed in a reagent card chamber of a special full-automatic urine analyzer, the sample size is 30ul, and the reaction time is 90 seconds.
(2) The standard solution of example 4 was placed in a sample pan of a fully automatic urine analyzer, and the standard solution was measured one by one to draw a calibration curve.
(3) When the device is used, a sample is placed in a sample disc of the full-automatic urine analyzer, and after the device is detected to be finished, the concentration of the sample is automatically calculated by the device according to the calibration curve.
Example 6: accuracy analysis
By using the test strips of examples 1 to 3, human serum albumin standards with concentrations of 10mg/L, 40mg/L and 160mg/L are detected according to the method of example 5, wherein the test strip of example 1 detects 40mg/L standard, the test strip of example 2 detects 10mg/L standard, and the test strip of example 3 detects 160mg/L standard, and the results are shown in table 1.
TABLE 1
The result shows that the relative deviation of the detection result of each test strip is less than 10% of the standard requirement, which indicates that the method has high accuracy, and particularly the relative deviation of the product of the embodiment 3 is less than 1%, and the accuracy is extremely high.
Example 7: precision analysis
Any urine sample was tested 10 times using the test strip of example 3 in the same manner as in example 5, and the results are shown in Table 2.
TABLE 2
The result shows that the standard deviation rate is 3.1 percent and is far less than 10 percent of the standard requirement, which indicates that the method has high precision. In addition, the standard deviation of the test strip of the embodiment 2-3 is 3.4% and 3.3%, respectively, and both do not exceed 3.5%.
Example 8: analysis of linear relationships
Using the test strip of example 3, a 320mg/L high albumin urine sample was used as a test sample, and the urine sample was diluted to 7 different concentrations, which were 0mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L, 160mg/L, and 320mg/L in this order, and each concentration of the above samples was tested 2 times using the test strip of example 3 and the test method of example 5, and the correlation coefficient R value was calculated, and the results are shown in Table 3.
TABLE 3
The result shows that the R value is 0.9999 and is close to 1, which indicates that the reagent and the test strip thereof have excellent linearity in the range of 0-320.0 mg/L. In addition, the test strips of examples 2-3 were used for linear analysis, and the R values were 0.9997 and 0.9999, respectively.
Example 9: comparative test with reference
1. Comparative test for control 1
On the basis of the test strip of the embodiment 3, a common methanol solvent is adopted to replace a toluene reagent in the solution B of the invention, and sodium molybdate and sucrose are removed at the same time to prepare a reference substance 1;
the test strip of example 3 and the control 1 were used to test the same sample by the test method of example 5, each sample was tested 10 times, and the test results are shown in table 4.
TABLE 4
The result shows that the CV value of the reference substance 1 exceeds 10% of the required CV value, which indicates that the precision of the methanol solvent optimized by the test strip of the invention is better than that of the chloroform solvent, and indicates that the method of the invention has good precision.
2. Comparative test for control 2
On the basis of the test paper strip of the embodiment 3, the citric acid in the solution B is transferred to the solution A, the whole sodium oxalate buffer solution is replaced, and a common methanol solvent is adopted to replace a toluene reagent in the solution B to prepare a reference substance 2;
for 20 samples with fixed values, the test strip of example 1 and the control 2 were used respectively to perform the detection method of example 6, each sample was tested 1 time, and the detection results are shown in table 5.
TABLE 5
The result shows that the test strip has good correlation with a fixed value sample, is superior to a reference substance 2 in both accuracy and precision, and shows that the method has high accuracy and precision.
Example 10: comparative test with existing semi-quantitative product
Comparison test of positive detection rate with Korean Saibao same methodology albumin test strip
Detecting a sample: a set of 20 urine samples; sample values at 30 mg/L and below were negative samples, and at 30 mg/L and above were positive samples.
20 samples were tested 1 time each using the product of example 3 and the test methods of examples 4-5, and the results are shown in Table 6, comparing with the test results of Korea Saibao.
TABLE 6
Note: the quantitative detection result of the invention is shown in brackets, and the semi-quantitative result is shown outside the brackets;
the result shows that the method is superior to Korea Saibao in positive detection rate, and shows that the method has strong anti-interference and low false positive rate.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
1. A reagent for detecting microalbumin in urine is characterized by comprising a solution A and a solution B, wherein the solution A is a sodium oxalate buffer solution, the sodium oxalate buffer solution comprises sodium oxalate, sodium molybdate and sucrose, the concentration of the sodium molybdate in the sodium oxalate buffer solution is 2.1-2.3% (w/v), the concentration of the sucrose is 1.2-1.3% (w/v), and the pH value of the sodium oxalate buffer solution is 3.0-3.4;
the solution B takes tetrahydrofuran and toluene as solvents, and contains 1.0-1.4% (w/v) of citric acid, 3.0-5.0% (v/v) of polypropylene glycol and 0.04-0.10% (w/v) of sulfophthalein dye, wherein the volume ratio of tetrahydrofuran to toluene is 1: (1-1.5).
2. Use of the reagent of claim 1 in the preparation of a test strip for the detection of microalbumin in urine.
3. A test strip for detecting urine microalbumin is characterized in that the test strip is prepared by soaking carrier filter paper in liquid A and liquid B of the reagent of claim 1 uniformly and then drying.
4. The test strip of claim 3, wherein the carrier filter paper is a 3MM chromatographic filter paper.
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| CN116699138A (en) * | 2020-12-03 | 2023-09-05 | 湖南优博思生物技术有限公司 | Semi-quantitative pepsin detection product for distinguishing physiological and pathological reflux |
| CN114034693A (en) * | 2021-11-04 | 2022-02-11 | 广州达安基因股份有限公司 | Combined test paper for detecting urine creatinine urine microalbumin semiquantitative, preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1165301A (en) * | 1996-03-01 | 1997-11-19 | 美国拜尔公司 | Improved method for detection of protein |
| CN1520517A (en) * | 2001-06-25 | 2004-08-11 | �����ι�˾ | Total protein detection methods and devices |
| CN103901034A (en) * | 2014-04-29 | 2014-07-02 | 中国科学院苏州生物医学工程技术研究所 | Detection reagent strip for detecting microalbumin in urine and preparation method of detection reagent strip |
| CN105388146A (en) * | 2015-10-20 | 2016-03-09 | 北京中生金域诊断技术股份有限公司 | Kit for simultaneously detecting sodium, creatinine and microalbumin in urine |
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- 2017-12-20 CN CN201711382361.7A patent/CN108120714B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1165301A (en) * | 1996-03-01 | 1997-11-19 | 美国拜尔公司 | Improved method for detection of protein |
| CN1520517A (en) * | 2001-06-25 | 2004-08-11 | �����ι�˾ | Total protein detection methods and devices |
| CN103901034A (en) * | 2014-04-29 | 2014-07-02 | 中国科学院苏州生物医学工程技术研究所 | Detection reagent strip for detecting microalbumin in urine and preparation method of detection reagent strip |
| CN105388146A (en) * | 2015-10-20 | 2016-03-09 | 北京中生金域诊断技术股份有限公司 | Kit for simultaneously detecting sodium, creatinine and microalbumin in urine |
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