CN108120714A - A kind of reagent for detecting urine microdose urine protein and its application - Google Patents
A kind of reagent for detecting urine microdose urine protein and its application Download PDFInfo
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Abstract
The present invention relates to technical field of medical detection, disclose a kind of reagent for detecting Urinary microalbumin and its application.Reagent of the present invention includes A liquid and B liquid, and the A liquid is sodium oxalate buffer solution, and the sodium oxalate buffer solution includes sodium oxalate and selected from one or both of sodium molybdate and sucrose；The B liquid includes citric acid, polypropylene glycol and phenolphthalein dyestuff using tetrahydrofuran and toluene as solvent.The present invention is by adjusting the component in optimization reagent, influence of each disturbing factor to detection in urine specimen is reduced, has reached higher accuracy and precision, and linear relationship is good in a wide range of, reliability is high, compensates for existing similar product insufficient in terms of accuracy and precision the defects of.
Description
Technical field
The present invention relates to technical field of medical detection, and in particular to it is a kind of detect urine microdose urine protein reagent and its
Using.
Background technology
Microdose urine protein is a kind of microalbumin occurred in urine, under normal circumstances the content pole in urine
It is few.The ability of kidney filtration albumin can be changed when lesion occurs for kidney, albumin is caused to leak into urine, therefore, urine
Microalbumin is the goldstandard of albuminuria.
At present, the detection mode of microdose urine protein has：Wet-chemical quantitatively detects, dry chemical qualitative detection and dry chemical half
Quantitative detection etc., wherein wet-chemical quantitatively detect it is different according to the detection time of different automatic clinical chemistry analyzers and take compared with
It is long；Dry chemical half-quantitative detection is then more suitable for scalping and looks into.
At present, detect in the market microalbumin test paper it is more well-known be South Korea match it is precious, but belong to sxemiquantitative
Detection method can only provide general content range, and cannot provide accurate content, and false positive is higher.
Chinese patent CN103901034A discloses a kind of reagent strip for detecting microdose urine protein, can both carry out half
Quantitative detection can also carry out quantitative detection, but since the selection of inappropriate reagent easily leads to the precision and standard of testing result
All deficiencies of exactness.
The content of the invention
In view of this, it is an object of the invention to provide a kind of reagents for detecting Urinary microalbumin so that the examination
Agent possesses higher accuracy and precision when detecting Urinary microalbumin；
Another object of the present invention is to provide the test strips based on mentioned reagent so that the test strips exist
Possess higher accuracy and precision when detecting Urinary microalbumin, reduce false positive results, while can quantify and half
Quantitative detection；
Another object of the present invention is to provide the quantitatively or semi-quantitatively detection method based on mentioned reagent, makes
It obtains the detection method and possesses higher accuracy and precision when detecting Urinary microalbumin.
To achieve these goals, the present invention provides following technical solution：
A kind of reagent for detecting Urinary microalbumin, including A liquid and B liquid, the A liquid is sodium oxalate buffer solution, described
Sodium oxalate buffer solution includes sodium oxalate and selected from one or both of sodium molybdate and sucrose；
The B liquid includes citric acid, polypropylene glycol and phenolphthalein dyestuff using tetrahydrofuran and toluene as solvent.
For existing detection Urinary microalbumin product insufficient in terms of accuracy and precision the defects of, this hair
The bright composition by adjusting detection reagent, precision and accuracy when significantly improving detection.
Preferably, the pH value of the sodium oxalate buffer solution is 3.0-3.4.It is described in the specific embodiment of the invention
The pH value of sodium oxalate buffer solution is adjusted by adding in the amount of sodium oxalate, such as oxalic acid na concn is 133g/1000mL, and pH value is
3.2。
Preferably, the concentration of sodium molybdate is 2.1%-2.3% (w/v) in the sodium oxalate buffer solution, the concentration of sucrose
For 1.2%-1.3% (w/v).In the specific embodiment of the invention, the concentration of the sodium molybdate is 2.1% or 2.3% (w/
V), the concentration of the sucrose is 1.2% or 1.3% (w/v)；More specifically, sodium molybdate is included in the sodium oxalate buffer solution,
Its concentration is 2.1% (w/v)；Or comprising sucrose in the sodium oxalate buffer solution, concentration is 1.2% (w/v)；Or the oxalic acid
Comprising sodium molybdate and sucrose in sodium buffer solution, molybdic acid na concn is 2.3% (w/v), and sucrose concentration is 1.3% (w/v)；In this hair
In bright, other interference components such as ion, urea etc. in urine is excluded by adding in sodium molybdate, and can be made by adding in sucrose
Reaction system diffusion during detection evenly, with this promotes the more accurate and accurate of testing result.
Preferably, the B liquid is using tetrahydrofuran and toluene as solvent, comprising 1.0%-1.4% (w/v) citric acid,
3.0%~5.0% (v/v) polypropylene glycol, 0.04%-0.10% (w/v) sulfonephthalein dyestuff.The present invention in conventional tetrahydrofuran and
On the basis of methanol, ethyl alcohol equal solvent, methanol or ethyl alcohol are replaced using toluene, more stable reaction is provided as reaction system
Environment reduces the influence to test result.
In the specific embodiment of the invention, the B liquid includes 1.0% (w/v) lemon using tetrahydrofuran and toluene as solvent
Lemon acid, 3.0% (v/v) polypropylene glycol, 0.04% (w/v) sulfonephthalein dyestuff；Or using tetrahydrofuran and toluene as solvent, comprising
1.2% (w/v) citric acid, 4.0% (v/v) polypropylene glycol, 0.06% (w/v) sulfonephthalein dyestuff；Or using tetrahydrofuran and toluene as
Solvent includes 1.3% (w/v) citric acid, 5.0% (v/v) polypropylene glycol, 0.08% (w/v) sulfonephthalein dyestuff；Preferably, institute
The volume ratio for stating hydrogen furans and toluene is 1：(1-1.5).
Meanwhile the invention also provides the reagent prepare detect Urinary microalbumin test strips in application or
Detect the application in Urinary microalbumin.
According to application the present invention provides a kind of test strips for detecting Urinary microalbumin, by carrier filter paper successively through institute
The A liquid and the even rear drying of B liquid leaching for stating reagent prepare.More specifically preparation process is as follows：
Carrier filter paper is soaked in A liquid even, suck redundant solution, dried in drying box, 100 DEG C of drying temperature, during drying
Between 30min；
The filter paper for being soaked with A liquid of drying in B liquid is soaked even, suck redundant solution, dried in drying box, drying temperature 60
DEG C, drying time 30min.
Operation and carrying for convenience, the test strips can cut into item, be pasted onto PVC on pieces, then cut into conjunction
The shape of suitable small item, the specification and final products can be adjusted arbitrarily, this depends on actual demand.
Preferably, the carrier filter paper is 3MM or 3 grade of chromatography filter paper.
According to the test strips of preparation, the present invention can also carry out urine sample detection quantitatively or semi-quantitatively, wherein half
Quantitative detection method is：
The bovine serum albumin solution of normal gradients concentration is added drop-wise to respectively in the test strips, obtains standard colorimetric
Card；
Urine sample is added drop-wise in the test strips, color and concentration on standard color comparison card are compared according to colour developing result
Correspondence, obtain semi-quantitative results.
More specifically, compound concentration be 320mg/L bovine serum albumin(BSA) standard solution, gradient dilution into 320mg/L,
7 concentration of 160mg/L, 80mg/L, 40mg/L, 20mg/L, 10mg/L, 0mg/L；The cow's serum of above-mentioned standard gradient concentration
Albumin solution is added in test strips, and sample size 30ul is observed after 90 seconds, can be visually observed out from colourless to navy blue bright
Colour developing rank, obtains standard color comparison card；This is loaded in test strips, sample size 30ul, compared with standard color comparison card, range estimation and sample
Block the concentration that the concentration corresponding to immediate standard color comparison card is sample to be tested.
And the method for quantitatively detecting microalbumin in urine includes：
The bovine serum albumin solution of normal gradients concentration is added drop-wise to respectively in the test strips, according to urinalysis
Instrument draws standard curve；
Urine sample is added drop-wise in the test strips, detected value is obtained by Urine Analyzer, according to standard curve
Obtain corresponding microalbumin concentration.
More specifically, the test strips are fitted into and special get stuck and in cartridge, be placed in dedicated full-automatic Urine Analyzer
Reagent card storehouse in, sample size 30ul, 90 seconds reaction time；
The bovine serum albumin solution of aforesaid standards gradient concentration is placed in the sample disk of full automatic urine analyzer, by
One detection, draws standard curve；
Sample is placed in the sample disk of full automatic urine analyzer, after to be detected, instrument according to standard curve from
It is dynamic to calculate surveyed concentration of specimens.
According to actually detected result of the test, the present invention has higher accuracy, and relative deviation is less than 5%, far below mark
The 10% of alignment request；Precision the results show standard rate is 3.1%, much smaller than the 10% of standard requirement, shows institute of the present invention
Method is stated with high precision；Linear relationship result of the test shows have in the range of 0.0mg/L-320.0mg/L good
The linearity, R values are 0.9999, close to 1.
In addition, the component in similar product is replaced on reagent of the present invention composition or deletes constituent part, composition control examination
Agent product is compared, the results show that bigger difference occur in the accuracy of reference product and precision, correlation R-value is
0.9347, CV value is more than the 10% of requirement.
From above technical scheme, the present invention reduces each in urine specimen by adjusting the component in optimization reagent
Influence of the disturbing factor to detection, has reached higher accuracy and precision, and linear relationship is good in a wide range of, reliably
Property it is high, compensate for existing similar product insufficient in terms of accuracy and precision the defects of.
Description of the drawings
Fig. 1 show standard color comparison card；Wherein, it is its corresponding concentration below color lump, unit mg/L.
Specific embodiment
The invention discloses a kind of reagents for detecting Urinary microalbumin and its application, those skilled in the art to borrow
Reflect present disclosure, is suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications are to this field
It is it will be apparent that they are considered as being included in the present invention for technical staff.Reagent of the present invention and its related application
Be described by preferred embodiment, related personnel substantially can not depart from present invention, in spirit and scope it is right
Reagent as described herein and its related application are modified or suitably change with combining, to realize and using the technology of the present invention.
Just a kind of reagent for detecting urine creatinine provided by the present invention and its application are described further below.
Embodiment 1：Reagent of the present invention and test strips
1st, reagent forms
A liquid is：133g sodium oxalates, 21g sodium molybdates (2.1%), purified water are settled to 1000mL, pH value 3.2.
B liquid is：10g citric acids (1.0%), 30mL polypropylene glycols (3.0%), 400mg phenolphthalein dyestuff (0.04%), with four
Hydrogen furans and toluene (volume ratio 1:1) it is settled to 1000mL.
2nd, the preparation of test strips
Filter paper is soaked in A liquid even, suck redundant solution, dried in drying box, 100 DEG C of drying temperature, drying time
30min；
The filter paper for being soaked with A liquid of drying in B liquid is soaked even, suck redundant solution, dried in drying box, drying temperature 60
DEG C, drying time 30min.
Dry filter paper cuts into item, is pasted onto PVC on pieces, then cuts into suitable small item.
Embodiment 2：Reagent of the present invention and test strips
1st, reagent forms
A liquid is：133g sodium oxalates, 12g sucrose (1.2%), purified water are settled to 1000mL, pH value 3.2.
B liquid is：12g citric acids (1.2%), 40mL polypropylene glycols (4.0%), 600mg phenolphthalein dyestuff (0.06%), with four
Hydrogen furans and toluene (volume ratio 1:1.5) it is settled to 1000mL.
2nd, the preparation of test strips
Filter paper is soaked in A liquid even, suck redundant solution, dried in drying box, 100 DEG C of drying temperature, drying time
30min；
The filter paper for being soaked with A liquid of drying in B liquid is soaked even, suck redundant solution, dried in drying box, drying temperature 60
DEG C, drying time 30min.
Dry filter paper cuts into item, is pasted onto PVC on pieces, then cuts into suitable small item.
Embodiment 3：Reagent of the present invention and test strips
1st, reagent forms
A liquid is：133g sodium oxalates, 23g sodium molybdates (2.3%), 13g sucrose (1.3%), purified water is settled to 1000mL,
PH value is 3.2.
B liquid is：13g citric acids (1.3%), 50mL polypropylene glycols (5.0%), 800mg phenolphthalein dyestuff (0.08%), with four
Hydrogen furans and toluene (volume ratio 1:1.5) it is settled to 1000mL.
2nd, the preparation of test strips
Filter paper is soaked in A liquid even, suck redundant solution, dried in drying box, 100 DEG C of drying temperature, drying time
30min；
The filter paper for being soaked with A liquid of drying in B liquid is soaked even, suck redundant solution, dried in drying box, drying temperature 60
DEG C, drying time 30min.
Dry filter paper cuts into item, is pasted onto PVC on pieces, then cuts into suitable small item.
Embodiment 4：Semi-quantitative detection method
(1) compound concentration be 320mg/L bovine serum albumin(BSA) standard solution, gradient dilution into 320mg/L, 160mg/L,
7 concentration of 80mg/L, 40mg/L, 20mg/L, 10mg/L, 0mg/L.
(2) above-mentioned standard liquid is added in test strips, sample size 30ul is observed after 90 seconds, can visually observed out from nothing
Color is to navy blue apparent color range.
(3) sample-adding is originally in test strips, and sample size 30ul, compared with standard color comparison card, range estimation is immediate with sample card
Concentration corresponding to standard color comparison card is the concentration of sample to be tested.Standard color comparison card is as shown in Figure 1.
Embodiment 5：Quantitative detecting method
(1) test strips are fitted into the special reagent card for getting stuck and in cartridge, being placed in dedicated full-automatic Urine Analyzer
In storehouse, sample size 30ul, 90 seconds reaction time.
(2) titer in embodiment 4 is placed in the sample disk of full automatic urine analyzer, detected one by one, it is fixed to draw
Mark song line.
(3) in use, sample is placed in the sample disk of full automatic urine analyzer, after to be detected, instrument according to
Calibration curve calculates surveyed concentration of specimens automatically.
Embodiment 6：Accuracy analysis
Using the test strips of embodiment 1-3, according to the method for embodiment 5 be respectively to concentration 10mg/L, 40mg/L,
The human serum albumins standard items of 160mg/L are detected, wherein, 1 ELISA test strip 40mg/L standard items of embodiment, embodiment
2 ELISA test strip 10mg/L standard items, 3 ELISA test strip 160mg/L standard items of embodiment, the results are shown in Table 1.
Table 1
The results show that each ELISA test strip result relative deviation of the present invention is respectively less than the 10% of standard requirement, show this hair
Bright the method has the product of high accuracy, particularly embodiment 3, and for relative deviation below 1%, accuracy is high.
Embodiment 7：Precision Analyze
Using the test strips of embodiment 3 to any one urine sample, detection 10 times is repeated according to 5 method of embodiment, as a result
It is shown in Table 2.
Table 2
The results show that standard rate is 3.1%, much smaller than the 10% of standard requirement, show that the method for the invention has
High precision.In addition, be detected according to the test strips of embodiment 2-3, standard deviation is respectively 3.4% and 3.3%, not less than
3.5%.
Embodiment 8：Linear relationship is analyzed
Using the test strips of embodiment 3, using the high albuminuria fluid samples of 320mg/L as detection sample, by urine sample
7 different concentration are diluted to, are followed successively by 0mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L, 160mg/L, 320mg/L,
2 detections are carried out to each concentration of above-mentioned sample using the test strips of embodiment 3 and the detection method of embodiment 5, calculate phase
Coefficients R value is closed, the results are shown in Table 3.
Table 3
The results show that R values are 0.9999, close to 1, show reagent of the present invention and its test strips 0-320.0mg/L's
In the range of have the splendid linearity.In addition, carrying out linear analysis using the test strips of embodiment 2-3, R values are respectively 0.9997
With 0.9999.
Embodiment 9：With the contrast test of reference substance
1st, the contrast test of reference substance 1
On the basis of the test strips of embodiment 3, the toluene reagent in B liquid of the present invention is replaced using common methanol solvate,
Sodium molybdate and sucrose are removed simultaneously, reference substance 1 is made；
Using the test strips of embodiment 3 and reference substance 1, by the detection method of embodiment 5, same sample is examined
It surveys, each pattern detection 10 times, testing result is shown in Table 4.
Table 4
The results show that the CV values of reference substance 1 have been over the 10% of requirement, show the preferred methanol of test strips of the present invention
Solvent precision is better than chloroform solvent, shows that the method for the invention precision is good.
2nd, the contrast test of reference substance 2
On the basis of the test strips of embodiment 3, the citric acid in B liquid of the present invention is moved in A liquid, replaces entire oxalic acid
Sodium buffer solution replaces the toluene reagent in B liquid of the present invention using common methanol solvate, reference substance 2 is made；
To 20 samples of definite value, the test strips of embodiment 1 and reference substance 2 is respectively adopted, passes through the inspection of embodiment 6
Survey method detects, and each pattern detection 1 time, testing result is shown in Table 5.
Table 5
The results show that test strips of the present invention and definite value sample correlations are good, no matter in accuracy or in precision
Upper better than reference substance 2, shows that the method for the invention accuracy and precision are all higher.
Embodiment 10：With the contrast test of existing sxemiquantitative product
Precious same methodology albumin test strips, which are matched, with South Korea does positive rate contrast test
Detect sample：20 urine specimens of definite value；Sample value is in 30mg/L and following for negative sample, in 30mg/L
It is positive sample above.
Using the detection method of 3 product of embodiment and embodiment 4-5 to 20 pattern detections, each pattern detection 1 time, with
The precious testing result of South Korea's match compares, and the results are shown in Table 6.
Table 6
Note：It is the quantitative testing result of the present invention in bracket, is semi-quantitative results outside bracket；
The results show that the method for the invention is precious better than South Korea's match on positive rate, show the method for the invention
Anti-interference strong, false positive rate is low.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of reagent for detecting Urinary microalbumin, which is characterized in that including A liquid and B liquid, the A liquid delays for sodium oxalate
Fliud flushing, the sodium oxalate buffer solution include sodium oxalate and selected from one or both of sodium molybdates and sucrose；
The B liquid includes citric acid, polypropylene glycol and phenolphthalein dyestuff using tetrahydrofuran and toluene as solvent.
2. reagent according to claim 1, which is characterized in that the pH value of the sodium oxalate buffer solution is 3.0-3.4.
3. reagent according to claim 1 or claim 2, which is characterized in that the concentration of sodium molybdate is in the sodium oxalate buffer solution
2.1%-2.3% (w/v), the concentration of sucrose is 1.2%-1.3% (w/v).
4. reagent according to claim 1, which is characterized in that the B liquid using tetrahydrofuran and toluene as solvent, comprising
1.0%-1.4% (w/v) citric acid, 3.0%~5.0% (v/v) polypropylene glycol, 0.04%-0.10% (w/v) sulfonephthalein dyestuff.
5. according to the reagent of claim 1 or 4, which is characterized in that the volume ratio of the tetrahydrofuran and toluene is 1：(1-
1.5)。
6. reagent described in claim 1-5 any one prepare detect Urinary microalbumin test strips in application or examining
Survey the application in Urinary microalbumin.
7. a kind of test strips for detecting Urinary microalbumin, which is characterized in that appointed successively through claim 1-5 by carrier filter paper
Drying prepares after the A liquid of one reagent of meaning and the leaching of B liquid are even.
8. test strips according to claim 7, which is characterized in that the carrier filter paper is 3MM or 3 grade of chromatography filter paper.
9. a kind of method of microalbumin in half-quantitative detection urine, which is characterized in that including：
The bovine serum albumin solution of normal gradients concentration is added drop-wise to respectively in the test strips described in claim 7 or 8, is obtained
Standard color comparison card；
Urine sample is added drop-wise in the test strips described in claim 7 or 8, face on standard color comparison card is compared according to colour developing result
The correspondence of color and concentration obtains semi-quantitative results.
A kind of 10. method for quantitatively detecting microalbumin in urine, which is characterized in that including：
The bovine serum albumin solution of normal gradients concentration is added drop-wise to respectively in the test strips described in claim 7 or 8, according to
Urine Analyzer draws standard curve；
Urine sample is added drop-wise in the test strips described in claim 7 or 8, detected value is obtained by Urine Analyzer, according to
Standard curve obtains microalbumin concentration in corresponding urine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711382361.7A CN108120714B (en) | 2017-12-20 | 2017-12-20 | Reagent for detecting microalbumin in urine and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711382361.7A CN108120714B (en) | 2017-12-20 | 2017-12-20 | Reagent for detecting microalbumin in urine and application thereof |
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| Publication Number | Publication Date |
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| CN108120714A true CN108120714A (en) | 2018-06-05 |
| CN108120714B CN108120714B (en) | 2020-11-27 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113049825A (en) * | 2020-12-03 | 2021-06-29 | 杨轶轩 | Semi-quantitative pepsin detection product for distinguishing physiological reflux from 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 |
-
2017
- 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113049825A (en) * | 2020-12-03 | 2021-06-29 | 杨轶轩 | Semi-quantitative pepsin detection product for distinguishing physiological reflux from 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108120714B (en) | 2020-11-27 |
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