CN109991177A - A kind of stabilization, bilirubin direct (enzymatic measurement) detection reagent of strong antijamming capability and detection method - Google Patents
A kind of stabilization, bilirubin direct (enzymatic measurement) detection reagent of strong antijamming capability and detection method Download PDFInfo
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Abstract
The present invention relates to serum direct bilirubin detection technique field, in particular to a kind of serum direct bilirubin (enzymatic measurement) detection reagent contains buffer in reagent R1, sodium fluoride, NAC (N-acetylcystein), 4- toluenesulfonic acid sodium salt, lipase, ascorbic acid oxidase, alpha-cyclodextrin, sucrose, trehalose, PEG-6000, fatty alcohol polyoxyethylene ether (AEO-9), preservative；Contain buffer, bilirubin oxidase, sucrose, trehalose, PEG-6000, fatty alcohol polyoxyethylene ether (AEO-9), preservative in reagent R2.This reagent significantly improves the specificity and accuracy when test bilirubin direct, can prevent reaction system muddiness, reaction is promoted to carry out, further increase the stability of reagent, anti-interference ability.
Description
Technical field
The present invention relates to serum direct bilirubin detection technique field, in particular to a kind of serum direct bilirubin detection examination
Agent further relates to the detection method using this detection reagent.
Background technique
Bilirubin is that the ferroheme deferrization released after red blood cell is destroyed is formed, and 80% bilirubin derives from spleen and liver,
Remaining bilirubin derives from marrow.Bilirubin is indirect bilirubin or unconjugated bilirubin, red in conjunction with gallbladder there are three types of existence form
Element, δ bilirubin.Wherein combine bilirubin and δ bilirubin that can directly react with diazo reagent, referred to as bilirubin direct.
The visible bilirubin direct content of various types of hepatitis, cirrhosis, obstructive jaundice increases, and compares transaminase
Sensitivity is particularly suitable for the diagnosis of chronic hepatitis evaluation of the prognosis and early-phase hepatocirrhosis.Direct gallbladder when severe anemia in serum is red
Cellulose content reduces.Therefore, the detection of serum bilirubin level has very important clinical meaning.
Currently, the detection method of bilirubin direct has diazo reagent method, high performance liquid chromatography, Bilirubin Oxidase method
Deng wherein diazo reagent method is easy to operate, but the method sensitivity and anti-hemolysis ability are poor；And high performance liquid chromatography, it is sensitive
Degree and specificity are preferable, but need specific apparatus, it is difficult to promote and apply；And Bilirubin Oxidase method, high sensitivity, not by haemolysis
It influences, but anti-interference ability and stability are poor.
In consideration of it, the present invention is on the basis of Bilirubin Oxidase method, by optimizing reaction system, using MES(2- (N-
Morpholine) ethanesulfonic acid) buffer, the steady of reagent can be effectively improved by adding stabilizer such as sucrose, trehalose, Macrogol 6000 etc.
It is qualitative；And alpha-cyclodextrin, lipase and ascorbic acid oxidase are added, it is possible to prevente effectively from the turbid interference with ascorbic acid of rouge,
Greatly enhance the anti-interference ability of reagent.And the bilirubin oxidase inhibitor fluorination of preferred dose is added in this law reagent
Sodium, NAC(N- acetylcysteine), 4- toluenesulfonic acid sodium salt, hence it is evident that specificity when improving test bilirubin direct and accurate
Property, furthermore it is preferred that the addition of new non-ionic surfactants fatty alcohol polyoxyethylene ether (AEO-9) can prevent reaction system
Muddy, promotion reaction carries out, and further increases the stability of reagent, anti-interference ability.The reagent operation is easy quickly, is applicable in
It is a kind of more stable, strong antijamming capability bilirubin direct (DBIL) detection reagent in automated analysis.
Summary of the invention
The object of the present invention is to provide a kind of reagent for being used to detect serum direct bilirubin (DBIL) and use the reagent
The method for detecting serum direct bilirubin.The kit uses enzymatic measurement, can effectively detect containing for serum direct bilirubin
Amount, has many advantages, such as that strong antijamming capability, stability are good.
Basic principle:
In the buffer that pH is 4.5 or so, when having inhibitor sodium fluoride, NAC(N- acetylcysteine), 4- toluenesulfonic acid sodium salt
In the presence of, the bilirubin direct in sample is optionally oxidized to biliverdin in bilirubin oxidase (BOD), by
The caused absorbance change of this reaction is measured under 450nm wavelength can acquire the content of bilirubin direct；
Bilirubin oxidase
Bilirubin direct bilirubinBiliverdin
PH=4.5 or so, inhibitor
What the present invention was obtained through the following steps:
A kind of serum direct bilirubin detection reagent, including reagent R1 and reagent R2, the reagent R1 and reagent R2 composition such as
Under:
Contain in reagent R1
Buffer························································50mmol/L,
Sodium fluoride························································2 mmol/L,
NAC(N- acetylcysteine)···········································0.2 mmol/L,
4- toluenesulfonic acid sodium salt··················································40 mmol/L,
Lipase························································15KU/L,
Ascorbic acid oxidase················································8 KU/L,
Alpha-cyclodextrin······················································1.5g/L
Sucrose··························································40g/L,
Trehalose························································10g/L,
PEG-6000······················································2g/L,
Fatty alcohol polyoxyethylene ether (AEO-9)·······································1g/L,
Preservative························································0.2mL/L；
2) component of reagent R2 are as follows:
Buffer························································50mmol/L,
Bilirubin oxidase··················································12KU/L,
Sucrose··························································40g/L,
Trehalose························································10g/L,
PEG-6000······················································2g/L,
Fatty alcohol polyoxyethylene ether (AEO-9)·······································1g/L,
Preservative························································0.2mL/L；
The serum direct bilirubin detection reagent, buffer is 25 DEG C in reagent R1, the MES buffer that pH is 4.5；
The serum direct bilirubin detection reagent, buffer is 25 DEG C in reagent R2, the MES buffer that pH is 4.5；
The serum direct bilirubin detection reagent, the preservative are Proclin300；
The serum direct bilirubin detection reagent detects the detection method of serum direct bilirubin content, using full-automatic
Biochemical Analyzer is measured using end-point method, and detection dominant wavelength is 450nm；
The ratio of the detection method, R1 reagent and R2 reagent is 3:1.
Beneficial effects of the present invention:
1) optimizing reaction system, using MES(2- (N- morpholine) ethanesulfonic acid) buffer, addition stabilizer for example sucrose, trehalose,
Macrogol 6000 etc. can effectively improve the stability of reagent；
2) lipase, alpha-cyclodextrin and ascorbic acid oxidase are added, it is possible to prevente effectively from the turbid interference with ascorbic acid of rouge, greatly
The anti-interference ability of big Contrast agent；
3) be added preferred dose bilirubin oxidase inhibitor sodium fluoride, NAC(N- acetylcysteine), 4- toluenesulfonic acid
Sodium, hence it is evident that improve the specificity and accuracy when test bilirubin direct；
4) addition of new non-ionic surfactants fatty alcohol polyoxyethylene ether (AEO-9) can prevent reaction system muddiness, promote
It is carried out into reaction, further increases the stability of reagent, anti-interference ability；
5) it the accuracy of reagent and has good stability, strong interference immunity is easy to use, can satisfy clinical needs completely.
Detailed description of the invention
Fig. 1 is the correlation curve figure of two kinds of reagents；
Fig. 2 is that two kinds of reagents imitate phase stability curve figure；
Fig. 3 is 1 reagent test method of embodiment；
Fig. 4 is that embodiment reagent interference free performance compares；
Fig. 5 is bilirubin direct (enzymatic measurement) the assay kit comparison that 1 reagent of embodiment is common with market and gets the nod
Testing result.
Specific embodiment
Invention is further explained combined with specific embodiments below:
Embodiment 1
The detection reagent of serum direct bilirubin, packet reagent R1 and reagent R2:
1) composition of its R1 are as follows:
MES(2- (N- morpholine) ethanesulfonic acid) buffer (pH=4.5,25 DEG C)··················50mmol/L,
Sodium fluoride·······················································2 mmol/L,
NAC(N- acetylcysteine)··········································0.2 mmol/L,
4- toluenesulfonic acid sodium salt·················································40 mmol/L,
Lipase·······················································15KU/L,
Ascorbic acid oxidase···············································8 KU/L,
Alpha-cyclodextrin·····················································1.5g/L
Sucrose·························································40g/L,
Trehalose·······················································10g/L,
PEG-6000·····················································2g/L,
Fatty alcohol polyoxyethylene ether (AEO-9)······································1g/L,
Preservative Proclin300·············································0.2mL/L；
2) component of reagent R2 are as follows:
MES(2- (N- morpholine) ethanesulfonic acid) buffer (pH=4.5,25 DEG C)··················50mmol/L,
Bilirubin oxidase·················································12KU/L,
Sucrose·························································40g/L,
Trehalose·······················································10g/L,
PEG-6000·····················································2g/L,
Fatty alcohol polyoxyethylene ether (AEO-9)······································1g/L,
Preservative Proclin300·············································0.2mL/L；
3) application method of the present embodiment reagent:
The serum direct bilirubin detection reagent of the present embodiment description, when in use using the full-automatic life with double reagent function
Change analyzer, such as 7180 fully-automatic analyzer of Hitachi, is measured using end-point method.R1 and R2 is put according to the ratio of 3:1
It sets on corresponding reagent position, places distilled water, standard items and sample, operation such as Fig. 3 in the corresponding position of sample disc.
Embodiment 2
Interference test: taking fresh mix serum, be divided into 2 equal portions, every equal portions are then separated into 7 equal portions, and different do is added
Substance is disturbed, its concentration in serum is made to reach the requirement of Fig. 4.Then 1 gained reagent of embodiment is used respectively, it is common simultaneously with market
The content of bilirubin direct in bilirubin direct (enzymatic measurement) reagent while comparative determination serum of approval, control group measurement knot
The measurement result of each group is shown in Fig. 4 after fruit and addition disturbance substance.Relative deviation (%)=(the measurement mean value-of interference sample is right
This measurement mean value in the same old way)/check sample measurement mean value × 100%.
As seen from Figure 4, hemoglobin≤90mg/dL, chylomicron≤2700 turbidity units, glucose in the sample
Survey when≤500mmol/L, ascorbic acid≤10mg/dL, urea≤17mmol/L, uric acid≤2 μm ol/L to 1 reagent of embodiment
Test result does not significantly interfere with.And group reagent is compareed in the presence of above-mentioned concentration interfering substance, it is significantly interfered with, this explanation is logical
Optimization reaction buffer system, addition lipase, alpha-cyclodextrin and ascorbic acid oxidase are crossed, and application new non-ionic surface is living
After property agent fatty alcohol polyoxyethylene ether (AEO-9), the interference free performance of 1 reagent of embodiment is significantly improved, far superior to having a competition
Agent.
Embodiment 3
Correlation experiment: being formulated reagent preparation using embodiment 1, and common State Food and Drug Administration is approved with market
Bilirubin direct (enzymatic measurement) detection kit of certain company carry out control test, while having detected 20 clinical serum samples
This, testing result is as shown in Figure 5.And the correlation curve (as shown in Figure 1) of two kinds of reagents is obtained, it is aobvious by testing result
Show, the related coefficient of two kits is 0.9996, illustrates that the two has great correlation.
Embodiment 4
The stability contrast of reagent is tested: to the reagent in embodiment 1, uniformly 13 groups of packing, every group of amount of reagent is that R1 is
18mL, R2 6mL；And the direct gallbladder for certain company that the State Food and Drug Administration for taking 13 groups of markets common is approved is red
Plain (enzymatic measurement) kit compares.It is placed into 2-8 DEG C of refrigerator, one group reagent of taking-up on the same day monthly detects DBIL matter
Control product (target value is 20.8 μm of ol/L), testing result is as shown in Fig. 2, 1 reagent of embodiment is more normal than market under 2-8 DEG C of condition of storage
Bilirubin direct (enzymatic measurement) assay kit seen is more stable.
By verifying, this reagent and similar detection reagent comparison correlation are good, and clinical detection sample results are consistent, Neng Gouda
To market to the application requirement of product, and good in anti-interference performance, it is a kind of more stable, good bilirubin direct (oxidation
Enzyme process) detection reagent.
Claims (7)
1. a kind of serum direct bilirubin (enzymatic measurement) detection reagent, it is characterised in that described including reagent R1 and reagent R2
The composition of reagent R1 and reagent R2 are as follows:
Contain in reagent R1
Buffer························································50mmol/L,
Sodium fluoride························································2 mmol/L,
NAC(N- acetylcysteine)···········································0.2 mmol/L,
4- toluenesulfonic acid sodium salt··················································40 mmol/L,
Lipase······· ······ ···········································15KU/L,
Ascorbic acid oxidase··············· · ································8 KU/L,
Alpha-cyclodextrin······················································1.5g/L
Sucrose··························································40g/L,
Trehalose························································10g/L,
PEG-6000······················································2g/L,
Fatty alcohol polyoxyethylene ether (AEO-9)·······································1g/L,
Preservative························································0.2mL/L；
2) component of reagent R2 are as follows:
Buffer························································50mmol/L,
Bilirubin oxidase··················································12KU/L,
Sucrose··························································40g/L,
Trehalose························································10g/L,
PEG-6000······················································2g/L,
Fatty alcohol polyoxyethylene ether (AEO-9)····· · ·································1g/L,
Preservative························································0.2mL/L。
2. serum direct bilirubin detection reagent according to claim 1, it is characterised in that buffer is 25 in reagent R1
DEG C, the MES buffer that pH is 4.5.
3. serum direct bilirubin detection reagent according to claim 1, it is characterised in that buffer is 25 in reagent R2
DEG C, the MES buffer that pH is 4.5.
4. serum direct bilirubin detection reagent according to claim 1, it is characterised in that surface is living in reagent R1 and R2
Property agent be fatty alcohol polyoxyethylene ether (AEO-9).
5. serum direct bilirubin detection reagent according to claim 1, it is characterised in that the preservative is
Proclin300。
6. a kind of detect the direct gallbladder of serum using the serum direct bilirubin detection reagent of any of claims 1-5
The detection method of red pigment, it is characterised in that be measured using automatic clinical chemistry analyzer using end-point method, detection dominant wavelength is
450nm。
7. detection method according to claim 6, it is characterised in that the ratio of R1 reagent and R2 reagent is 3:1.
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Cited By (5)
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| CN110687306A (en) * | 2019-10-30 | 2020-01-14 | 深圳上泰生物工程有限公司 | Direct-connection on-board hemolytic enzyme method double-reagent glycosylated hemoglobin detection kit |
| CN112710854A (en) * | 2021-01-08 | 2021-04-27 | 中拓生物有限公司 | Anti-interference and stable serum total bilirubin (enzyme method) determination kit and preparation method and application thereof |
| CN112710853A (en) * | 2021-01-08 | 2021-04-27 | 中拓生物有限公司 | Anti-interference and stable serum direct bilirubin (enzyme method) determination kit and preparation method and application thereof |
| CN113049839A (en) * | 2019-12-27 | 2021-06-29 | 桂林英美特生物技术研究所 | Stable liver function composite quality control product |
| CN114058676A (en) * | 2020-07-31 | 2022-02-18 | 北京九强生物技术股份有限公司 | Anti-factor Xa activity determination kit |
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| CN112710853A (en) * | 2021-01-08 | 2021-04-27 | 中拓生物有限公司 | Anti-interference and stable serum direct bilirubin (enzyme method) determination kit and preparation method and application thereof |
| CN112710853B (en) * | 2021-01-08 | 2021-11-02 | 中拓生物有限公司 | Anti-interference and stable serum direct bilirubin (enzyme method) determination kit and preparation method and application thereof |
| CN112710854B (en) * | 2021-01-08 | 2021-11-23 | 中拓生物有限公司 | Anti-interference and stable serum total bilirubin (enzyme method) determination kit and preparation method and application thereof |
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Application publication date: 20190709 |