CN105548171B - A kind of cerebrospinal fluid and Urine proteins liquid single-reagent and preparation method thereof - Google Patents
A kind of cerebrospinal fluid and Urine proteins liquid single-reagent and preparation method thereof Download PDFInfo
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- CN105548171B CN105548171B CN201610014684.XA CN201610014684A CN105548171B CN 105548171 B CN105548171 B CN 105548171B CN 201610014684 A CN201610014684 A CN 201610014684A CN 105548171 B CN105548171 B CN 105548171B
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Abstract
The present invention provides a kind of cerebrospinal fluid and Urine proteins liquid single-reagent detection kit and preparation method thereof, belongs to external diagnosis reagent field.It is low to solve existing cerebrospinal fluid and Urine proteins liquid single-reagent detection kit precision and stability, the problem of reagent contamination instrument etc..The kit is made of single reagent.The present invention also provides a kind of cerebrospinal fluid and the preparation method of Urine proteins liquid single-reagent detection kit.Dmso solution pyrogallol red is used in the reagent, before dissolving, TritonX 305 is added in dimethyl sulfoxide, pyrogallol red is added, reagent can be effectively eliminated and glue cuvette, avoid cuvette from polluting, and pyrogallol red is uniformly dispersed, improve sensitivity and the precision of reagent.2 formyl chloride-1s are added in the reagent, 1 dimethylhydrazine, makes the bluish violet compound color that pyrogallol red molybdenum is formed with protein substantially deepen, and improves reagent sensitivity.
Description
Technical field
The present invention provides a kind of cerebrospinal fluid and Urine proteins liquid single-reagent and preparation method thereof, belongs to and is related to in-vitro diagnosis examination
Agent field.
Background technology
Common cerebrospinal fluid and urinary proteins analysis method has pyrogallol red molybdenum method, biuret method and sulfo group currently on the market
Salicylic acid turbidimetry.Biuret be by the compound that forms of two molecule Ureas condensation, biuret in alkaline conditions with sulphur
Sour copper generates aubergine complex, contains peptide bond in protein molecule, similar with biuret structure, therefore protein and basic sulfate
Copper can also generate aubergine complex, its shade is directly proportional to protein concentration under certain condition, can be used to protein
It is quantitative.But the method no matter manual method or kit, deviation big the problems such as higher all there are measured value when measuring low value, especially
It is low with the method accuracy in detection since protein content is about 10-140mg/L in random urine when measuring random urine, and urinate
Sample dosage is big.
Sulfosalicylic acid turbidimetry is under the conditions of the pH of slightly below isoelectric points of proteins, and protein carries the ammonia of positive charge
Base is closed with negatively charged sulfosalicylic acid root knot, is formed insoluble protein salt and is precipitated.The change of its turbidity is sunk with acidity
The factors influences such as the concentration of the chemical property of shallow lake agent, temperature and acid measure, and interference free performance is poor, be generally used for it is qualitative, very
It is few to be used to quantify.
Kit currently on the market is all inadequate there are accuracy, and negative value occurs in low value test, and reaction product viscosity is big,
Cuvette easily is stained with, causes instrumental pollution, lowering apparatus service life.
The content of the invention
The present invention provides a kind of cerebrospinal fluid and Urine proteins liquid single-reagent, there is provided one kind can be in automatic clinical chemistry analyzer
Upper applicable cerebrospinal fluid and quantity of proteinuria detection kit, cerebrospinal fluid of the invention and quantity of proteinuria detection kit pass through
Different from the medicine and preparation method of the prior art, make the reagent more stable, dispersiveness is more preferable, and detection is more accurate, and will not be dirty
Dye damage instrument, the range of linearity reach 2-3000mg/L.
The present invention provides a kind of preparation method of cerebrospinal fluid and Urine proteins liquid single-reagent, suitable for industrialized production.
A kind of cerebrospinal fluid and Urine proteins liquid single-reagent of the present invention, it is characterised in that be by following medicine by weight
Made of portion rate：
Succinic acid-sodium benzoate buffer solution：50mmol/L, pyrogallol red：0.01g-0.1g, dimethyl sulfoxide：20-100
ml、TritonX-305：5-20 ml, sodium oxalate：1.39-5.56 g, 2- formyl -1,1- dimethylhydrazines：0.09-0.54g.
The present invention provides a kind of preparation method of cerebrospinal fluid and Urine proteins liquid single-reagent, comprises the following steps：
Weigh 5.94g succinic acid to be added in 1L purified waters, stirring and dissolving, add sodium benzoate 0.5g, stirring and dissolving,
HCl adjusts pH to 2.0-3.5 and is used as buffer solution；TritonX-305 5-20ml are measured to be added in 20-100ml dimethyl sulfoxides,
Mixing is sufficiently stirred, adds 0.01-0.1g pyrogallol reds, is stirred to being completely dissolved, is added to succinic acid-sodium benzoate and delays
In fliud flushing, 0.012-0.0484g sodium molybdates, 1.39-5.56g sodium oxalates, 0.09-0.54g 2- formyls -1,1- are sequentially added
Dimethylhydrazine.
Solvent used in the present invention is dimethyl sulfoxide, and appropriate TritonX-305, two kinds of medicines are added in dimethyl sulfoxide
Synergistic effect, contributes to the dissolving of pyrogallol red, plays an important role to the homogeneity and stability of reagent, be effectively improved survey
Try the accuracy of low value.
After 2- formyl -1,1- dimethylhydrazines are added, the color change of reagent reacting becomes apparent from, and further improves reagent spirit
Sensitivity, improves linear measured value and reaches the standard grade 30%.
The positive effect of the present invention is：
Pyrogallol red combines to form red complex with molybdic acid, has the absorption of maximum in 467nm, this compound is in acidity
Under the conditions of with protein binding formed bluish violet compound, have under 598nm maximum absworption peak, by the peak value for measuring 598nm
Even if can protein concentration.Pyrogallol red is insoluble in water, commonly uses the dissolving of the organic solvents such as methanol, but the organic solvent such as methanol dissolves
Pyrogallol red there is larger viscosity, and pyrogallol red disperses uneven, is tested on automatic clinical chemistry analyzer easy
There is exceptional value, and viscous cuvette, cause cuvette to pollute.
Using dmso solution pyrogallol red, before dissolving, TritonX-305 is added in dimethyl sulfoxide, then add
Enter pyrogallol red, reagent can be effectively eliminated and glue cuvette, and improve the sensitivity of reagent, there is preferably repeatability, survey
When low value urine sample and cerebrospinal fluid, deviation is small, suitable for automatic clinical chemistry analyzer.The advantages of pyrogallol red molybdenum method, is reagent
Stablize after preparation, can preserve for a long time.Kit prepared by the present invention, after 2- formyl -1,1- dimethylhydrazines are added, adjacent benzene three
The bluish violet compound color that phenol red molybdenum is formed with protein substantially deepens so that measured value is sensitiveer, is linearly higher by than before
30%。
Brief description of the drawings
Fig. 1 is linear measured value scatter diagram of the embodiment 1 to embodiment 3.
Embodiment
By following embodiments further illustrate description the present invention, do not limit the invention in any way, without departing substantially from
On the premise of the technical solution of the present invention, easy to implement any of those of ordinary skill in the art made for the present invention changes
Dynamic or change is fallen within scope of the presently claimed invention.
Embodiment 1：
Weigh 5.94g succinic acid to be added in 1L purified waters, stirring and dissolving, add sodium benzoate 0.5g, stirring and dissolving,
HCl adjusts pH to 2.0（25℃）It is spare as succinic acid-sodium benzoate buffer solution.TritonX-305 5ml are measured to be added to
In 20ml dimethyl sulfoxides, mixing is sufficiently stirred, adds 0.01g pyrogallol reds, stirs to being completely dissolved, is added to fourth two
In acid-sodium benzoate buffer solution, 0.012g sodium molybdates, 1.39g sodium oxalates, 0.09g 2- formyl -1,1- diformazans are sequentially added
Base hydrazine.
Embodiment 2
Weigh 5.94g succinic acid to be added in 1L purified waters, stirring and dissolving, add sodium benzoate 0.5g, stirring and dissolving,
HCl adjusts pH to 2.6（25℃）It is spare as succinic acid-sodium benzoate buffer solution.TritonX-305 10ml are measured to be added to
In 50ml dimethyl sulfoxides, mixing is sufficiently stirred, adds 0.05g pyrogallol reds, stirs to being completely dissolved, is added to fourth two
In acid-sodium benzoate buffer solution, 0.024g sodium molybdates, 2.78g sodium oxalates, 0.27g 2- formyl -1,1- diformazans are sequentially added
Base hydrazine.
Embodiment 3
Weigh 5.94g succinic acid to be added in 1L purified waters, stirring and dissolving, add sodium benzoate 0.5g, stirring and dissolving,
HCl adjusts pH to 3.5（25℃）It is spare as succinic acid-sodium benzoate buffer solution.TritonX-305 20ml are measured to be added to
In 100ml dimethyl sulfoxides, mixing is sufficiently stirred, adds 0.1g pyrogallol reds, stirs to being completely dissolved, is added to fourth two
In acid-sodium benzoate buffer solution, 0.0484g sodium molybdates, 5.56g sodium oxalates, 0.54g 2- formyl -1,1- diformazans are sequentially added
Base hydrazine.
The positive effect of the present invention place is proved by following comparative example：
Comparative example 1：
Following component reagent is prepared, for detection parameters with embodiment 1, preparation steps are as follows：
Weigh 5.94g succinic acid to be added in 1L purified waters, stirring and dissolving, add sodium benzoate 0.5g, stirring and dissolving,
HCl adjusts pH to 2.6（25℃）It is spare as succinic acid-sodium benzoate buffer solution.In 50ml dimethyl sulfoxides, 0.05g is added
Pyrogallol red, stirs to being completely dissolved, is added in succinic acid-sodium benzoate buffer solution, sequentially adds TritonX-
30510ml, 0.024g sodium molybdate, 2.78g sodium oxalates, 0.27g 2- formyl -1,1- dimethylhydrazines.
Comparative example 2：
Following component reagent is prepared, for detection parameters with embodiment 1, preparation steps are as follows：
Weigh 5.94g succinic acid to be added in 1L purified waters, stirring and dissolving, add sodium benzoate 0.5g, stirring and dissolving,
HCl adjusts pH to 2.6（25℃）It is spare as succinic acid-sodium benzoate buffer solution.TritonX-305 10ml are measured to be added to
In 50ml dimethyl sulfoxides, mixing is sufficiently stirred, adds 0.05g pyrogallol reds, stirs to being completely dissolved, is added to fourth two
In acid-sodium benzoate buffer solution, 0.024g sodium molybdates, 2.78g sodium oxalates are sequentially added.
Experiment in relation to cerebrospinal fluid and Urine proteins liquid list detection kit accuracy
Automatic biochemical analyzer parameter is set according to the requirement of each embodiment 1, embodiment 2, embodiment 3 and comparative example, use is bright
Road calibration object calibration, detects Landau quality-control product concentration, and each quality-control product is surveyed 3 times.CSF Concentration Testings the results are shown in Table 1.
Table 1
From table 1 it follows that embodiment measured value than comparative example measured value closer to target value, when be added without 2- formyls-
During 1,1- dimethylhydrazine, measured value is substantially relatively low, and the dissolving of pyrogallol red is improper that measured value can be caused higher, and poor repeatability, says
Bright kit measurement cerebrospinal fluid and Urine proteins ratio available reagent have more preferable accuracy.
After calibration, measure same urine specimen, survey 10 times, the concentration results by detecting 10 times try to achieve average value,
SD values, calculate CV values.It the results are shown in Table 2.
Table 2
It can be seen that from 2 data of table, embodiment 1-3 is significantly better than comparative example 1- to the measurement result of same sample, repeatability
2, when being added without 2- formyl -1,1- dimethylhydrazines, CV values reach 19.73%, illustrate that this reagent measure sample has than available reagent
There is more preferable accuracy, and can be seen that with reference to Tables 1 and 2, TritonX-305 additions reagent accuracy highest in 10ml.
The linear sample of autogamy is measured, the bovine serum albumin(BSA) of 3g/L is diluted to 8 concentration, each concentration mensuration knot is shown in
Table 3.
Table 3
The data of table 3 and the result of Fig. 1 can be seen that the measured value that embodiment is linearly reached the standard grade is higher than comparative example, reaches
More than 300mg/dl, related coefficient linearly improve 30% more than 0.9999, hence it is evident that better than comparative example.
Although the present invention is disclosed as above with embodiment, it is not limited to protection scope of the present invention, any ripe
The technical staff of this technology is known, in the change and retouching done without departing from the spirit and scope of the invention, this should all be belonged to
The scope of invention.
Claims (4)
- A kind of 1. liquid single-reagent detection kit for detecting cerebrospinal fluid and Urine proteins, it is characterised in that the kit by Single reagent R is formed, and R includes following chemical composition：
- 2. a kind of liquid single-reagent detection kit for detecting cerebrospinal fluid and Urine proteins according to claim 1, its feature It is, the buffer solution, selected from succinic acid-sodium benzoate buffer solution.
- 3. a kind of liquid single-reagent detection kit for detecting cerebrospinal fluid and Urine proteins according to claim 1, its feature It is, the buffer solution, pH 2.0-3.5.
- A kind of 4. preparation side of liquid single-reagent detection kit for detecting cerebrospinal fluid and Urine proteins according to claim 1 Method, it is characterised in that including：Step 1, first add pure water in reaction vessel, then sequentially adds succinic acid, sodium benzoate, adjusts pH with 4M HCl and arrive 2.0-3.5 is as buffer solution；Step 2, measurement TritonX-305 are added in dimethyl sulfoxide, are sufficiently stirred mixing, are added pyrogallol red, stir To being completely dissolved；Step 3, the mixed solution for obtaining step 2 are added in the mixed solution that step 1 obtains, sequentially add sodium molybdate, Sodium oxalate, 2- formyl -1,1- dimethylhydrazines, stir evenly.
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| CN106990102A (en) * | 2016-12-06 | 2017-07-28 | 山东大学齐鲁医院 | A kind of automatic clinical chemistry analyzer urine cerebrospinal fluid total protein detection reagent |
Citations (3)
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|---|---|---|---|---|
| CN102680701A (en) * | 2012-04-27 | 2012-09-19 | 嘉兴九七九生物技术有限公司 | Quantitative detection reagent and quantitative detection method for urinary protein liquid |
| CN104215604A (en) * | 2014-09-16 | 2014-12-17 | 中华人民共和国南通出入境检验检疫局 | Method for measuring content of proteins in grains and feed |
| CN104412097A (en) * | 2012-04-27 | 2015-03-11 | 生物辐射实验室股份有限公司 | Stain-free protein quantification and normalization |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2451611A1 (en) * | 2001-06-25 | 2003-01-03 | Bayer Healthcare Llc | Total protein detection methods and devices at low ph |
| JP2011013117A (en) * | 2009-07-02 | 2011-01-20 | Institute Of Physical & Chemical Research | Detection method and detection reagent of vpr protein |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680701A (en) * | 2012-04-27 | 2012-09-19 | 嘉兴九七九生物技术有限公司 | Quantitative detection reagent and quantitative detection method for urinary protein liquid |
| CN104412097A (en) * | 2012-04-27 | 2015-03-11 | 生物辐射实验室股份有限公司 | Stain-free protein quantification and normalization |
| CN104215604A (en) * | 2014-09-16 | 2014-12-17 | 中华人民共和国南通出入境检验检疫局 | Method for measuring content of proteins in grains and feed |
Non-Patent Citations (1)
| Title |
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| 邻苯三酚红钼络合显色法改良测定微量总蛋白;费春荣等;《浙江大学学报(医学版)》;19990228;第28卷(第01期);34-39页 * |
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