CN108181250A - A kind of heavy nitrogen total bilirubin detection reagent box of stabilization - Google Patents
A kind of heavy nitrogen total bilirubin detection reagent box of stabilization Download PDFInfo
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- CN108181250A CN108181250A CN201711427129.0A CN201711427129A CN108181250A CN 108181250 A CN108181250 A CN 108181250A CN 201711427129 A CN201711427129 A CN 201711427129A CN 108181250 A CN108181250 A CN 108181250A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/775—Indicator and selective membrane
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Abstract
At present, the stability of traditional heavy nitrogen total bilirubin detection reagent box there are it is certain the defects of, be mainly shown as, under conditions of not offset detection result 5% or so, can only generally control at 5 days or so or even can only control at 23 days.A kind of heavy nitrogen total bilirubin detection reagent box of stabilization of the present invention is by having mixed D glucopyranosides, D glucopyranoses, the 1-hydroxy ethylidene-1,1-diphosphonic acid stabilizer different with bextran 45 kind, and a cystic structures are formed, unstable material is coated with, when reacting, it is stimulated by extraneous active principle, it is discharged, so as to the stability of effective Contrast agent, there is higher marketing application value.
Description
Technical field
The present invention relates to clinical vitro detection reagent technique field, more particularly to a kind of total courage detected in serum or blood plasma
The detection kit of lycopene content.
Background technology
Bilirubin includes two kinds of unconjugated bilirubin and total bilirubin：Total bilirubin can be described as combining bilirubin, i.e. blood again
After middle unconjugated bilirubin transports liver, absorbed by liver cell rapidly, bilirubin in liver cell endochylema mainly with Y albumen and
Z protein bindings（Based on Y albumen）.Then it is water-soluble with reference to bilirubin that glucuronic acid generation is combined in endoplasmic reticulum；Trip
It can also become unconjugated bilirubin from bilirubin, i.e., not combined with glucuronic acid, unbonded bilirubin is mainly blood
Red pigment forms the water-soluble courage of linear tetrapyrrole under the effect catalysis of mononuclear phagocyte system cells microsome heme oxygenase
Green element, in cell liquid under the stronger biliverdin reductase effect catalysis of activity, biliverdin is further reduced into bilirubin.
At present for there are many kinds of the checkout and diagnosis methods of total bilirubin, mainly including heavy nitrogen, high performance liquid chromatography
（HPLC）, enzymatic measurement, vanadate method etc..Heavy nitrogen is a kind of total bilirubin detection method used earliest, have it is at low cost,
Using it is wide the characteristics of, but traditional heavy nitrogen is influenced by many factors, and result difference is larger；High performance liquid chromatography
（HPLC）It is more demanding to instrument and equipment, therefore conventional method should not be used as to promote and apply；Enzymatic measurement high sensitivity resists dry
Disturb it is indifferent, mainly since enzyme is high to temperature requirement, activity can only in certain temperature range competence exertion most fine piece of writing
With, and this kind of method reagent price is high, it is difficult to it promotes；Vanadate method is one proposed by Japanese scholars the nineties in last century
The novel method for oxidation of kind, but this method is larger to apparatus damage, the oxidisability of vanadate is unfavorable for protecting higher than potassium permanganate
It deposits.
In view of problem above, the present invention provides a kind of heavy nitrogen total bilirubin detection reagent boxes of stabilization.The reagent
Reagent in box is to be mixed by plurality of stable agent and protective agent according to different proportion, is effectively improved reagent and is transporting and storing
The unstability caused by environmental factor in the process provides more stable kit so as to detect bilirubin for heavy nitrogen.
Invention content
It is an object of the present invention to provide a kind of heavy nitrogen total bilirubin detection reagent box of stabilization, the invention kit emphasis solutions
Determined heavy nitrogen detection, storage and transportational process present in unstability and linear problem.
Basic principle
Total bilirubin and 2,4- dichloroaniline diazol form diazonium compound after combining, which is presented in acid condition
Go out red, cause the variation of the absorption photometric value at 545nm, the content of diazonium compound can be counted by absorption photometric variation
It calculates, and then calculates the content of total bilirubin in serum.
It is as follows using total bilirubin content method in kit measurement serum of the present invention：Detection wavelength is 545nm, sample
Amount：Reagent R1：Reagent R2=10ul：200ul：50ul.
The present invention is a kind of heavy nitrogen total bilirubin detection reagent box of stabilization, and the kit is to include reagent R1 and examination
Agent R2, ingredient and concentration are as follows：
Reagent R1 is：
Disodium ethylene diamine tetraacetate 0.1%-0.5%
Aminobenzenesulfonic acid 50mmol/L-100mmol/L
Sodium chloride 5g/L-9g/L
D- glucopyranosides 0.05%-0.1%
D- glucopyranoses 0.075%-0.2%
Glucan 0.3%-0.5%
Triton X-405 0.125%-0.4%
Citric acid-glycine buffer 0.1mol/L
Reagent R2 is：
2,4 dichloro aniline diazol 50mmol/L-100mmol/L
Ethylenediamine tetra-acetic acid 0.1%-1%
Hydrochloric acid 0.1mol/L-0.4mol/L
Glycine-HCI buffer solution 0.1mol/L
D- glucopyranosides 0.05%-0.1%
D- glucopyranoses 0.075%-0.2%
Glucan 0.3%-0.5%
1-hydroxy ethylidene-1,1-diphosphonic acid 10mmol/L-20mmol/L.
A kind of heavy nitrogen total bilirubin detection reagent box of stabilization of kit of the present invention includes reagent R1 and R2, wherein described
Each substance selection gist is as follows in reagent R1：
Disodium ethylene diamine tetraacetate is a kind of ion chelating agent, it can effective chelated metal ions, prevent some foreign ions
Unnecessary interference is caused to detection reaction；
Aminobenzenesulfonic acid is as color generation, for making azo dyes；
For simulating blood environment in human body, ensure to react carried out under the same terms in vitro sodium chloride, so as to
Ensure the authenticity and reliability of detection；
D- glucopyranosides, D- glucopyranoses, glucan are as stabilizer substance；
Triton X-405 are up to more than 15 surfactant as HLB, and effect substantially improves place of the reagent to sample
Rationality energy, the effective detection accuracy for ensureing reagent；
Citric acid-glycine buffer is mainly cushioning effect, ensures the stabilization of external environment during reaction, causes testing result will not
It is fluctuated and is influenced.
Each substance selection gist is as follows in the reagent R2：
2,4- dichloroaniline diazols are combined to form diazol with total bilirubin as reaction substrate, in the presence of azo dyes,
Red is presented, causes the variation of absorption photometric value at 545nm, containing for total bilirubin is calculated by the variation of absorption photometric value indirectly
Amount；
Disodium ethylene diamine tetraacetate is a kind of ion chelating agent, it can effective chelated metal ions, prevent some foreign ions
Unnecessary interference is caused to detection reaction；
Hydrochloric acid provides a kind of strong acidic environment；
Glycine-HCI buffer solution is mainly cushioning effect, ensures the stabilization of external environment during reaction, causes testing result will not be by
To fluctuation and influence；
D- glucopyranosides, D- glucopyranoses, glucan and 1-hydroxy ethylidene-1,1-diphosphonic acid are as stabilizer substance.
It is automated in full-automatic analyzer present invention can apply to series such as enlightening is auspicious, Mai Rui, Hitachi, Toshiba
Detection.
Using the present invention carry out in vitro sample diagnosis detection when, operating method is as follows, setting wavelength be 545nm, end-point method,
Sample size：Reagent R1：Reagent R2=10ul：200ul：50ul.Specimen needle is drawn sample and is incubated after in reagent R1, to protect
The stabilization of reaction environment external condition is demonstrate,proved, it is Δ A1 then to read absorption photometric value, and then adds in reagent R2, total courage in sample
Red pigment and 2,4- dichloroaniline diazol, which combine, forms diazol, and diazol shows red with aminobenzenesulfonic acid generation color reaction
Color causes the variation of absorption photometric value at wavelength 545nm, and it is Δ A2 that absorption photometric value is read in 37 DEG C of reactions, which for 5-10 minutes or so,
By calculating the variation of absorption photometric value, and then calculate the content of total bilirubin.Calculation formula is as follows：
Total bilirubin content=concentration of standard solution * absorption photometric values（ΔA2-ΔA1）.
Relative to the heavy nitrogen total bilirubin detection reagent box that existing market circulates, stabilization of kit of the present invention is preferable,
And precision is high, reproducible, strong antijamming capability, suitable for automatic clinical chemistry analyzer, Clinical practice value is larger.
Description of the drawings
Fig. 1 is reagent of the present invention and contrast agents Detection of Stability result.
Fig. 2 is kit of the present invention and contrast agents box interference--free experiments result.
Fig. 3 is kit of the present invention and contrast agents box clinical sample testing result.
Fig. 4 is kit of the present invention and contrast agents box clinical correlation collection of illustrative plates.
Specific embodiment
Below in conjunction with figure, the present invention will be described for embodiment.
Embodiment 1
In specific implementation, the reagent R1 is the present invention：
Disodium ethylene diamine tetraacetate 0.1%-0.5%
Aminobenzenesulfonic acid 50mmol/L-100mmol/L
Sodium chloride 5g/L-9g/L
D- glucopyranosides 0.05%
D- glucopyranoses 0.075%
Glucan 0.3%
Triton X-405 0.125%-0.4%
Citric acid-glycine buffer 0.1mol/L
Reagent R2 is：
2,4 dichloro aniline diazol 50mmol/L-100mmol/L
Ethylenediamine tetra-acetic acid 0.1%-1%
Hydrochloric acid 0.1mol/L-0.4mol/L
Glycine-HCI buffer solution 0.1mol/L
D- glucopyranosides 0.05%
D- glucopyranoses 0.075%
Glucan 0.3%
1-hydroxy ethylidene-1,1-diphosphonic acid 20mmol/L.
Embodiment 2
Reagent R1 is：
Disodium ethylene diamine tetraacetate 0.1%-0.5%
Aminobenzenesulfonic acid 50mmol/L-100mmol/L
Sodium chloride 5g/L-9g/L
D- glucopyranosides 0.075%
D- glucopyranoses 0.125%
Glucan 0.4%
Triton X-405 0.125%-0.4%
Citric acid-glycine buffer 0.1mol/L
Reagent R2 is：
2,4 dichloro aniline diazol 50mmol/L-100mmol/L
Ethylenediamine tetra-acetic acid 0.1%-1%
Hydrochloric acid 0.1mol/L-0.4mol/L
Glycine-HCI buffer solution 0.1mol/L
D- glucopyranosides 0.05%
D- glucopyranoses 0.075%
Glucan 0.3%
1-hydroxy ethylidene-1,1-diphosphonic acid 15mmol/L.
Embodiment 3
Reagent R1 is：
Disodium ethylene diamine tetraacetate 0.1%-0.5%
Aminobenzenesulfonic acid 50mmol/L-100mmol/L
Sodium chloride 5g/L-9g/L
D- glucopyranosides 0.1%
D- glucopyranoses 0.2%
Glucan 0.5%
Triton X-405 0.125%-0.4%
Citric acid-glycine buffer 0.1mol/L
Reagent R2 is：
2,4 dichloro aniline diazol 50mmol/L-100mmol/L
Ethylenediamine tetra-acetic acid 0.1%-1%
Hydrochloric acid 0.1mol/L-0.4mol/L
Glycine-HCI buffer solution 0.1mol/L
D- glucopyranosides 0.075%
D- glucopyranoses 0.125%
Glucan 0.5%
1-hydroxy ethylidene-1,1-diphosphonic acid 10mmol/L.
Kit performance detection experiment of the present invention
Stability test detects
With real kit of the invention（Embodiment 1）It is compared, is calibrated with conventional heavy nitrogen total bilirubin detection reagent box
Liquid and quality-control product are Landau 926UN and 1005UN respectively, the use of instrument are automatic clinical chemistry analyzer BS-800, parameter setting is such as
Under, wavelength 545nm, the Direction of Reaction is positive direction, and reading point is set as 14-16 points and 31-33 points, sample size：Reagent R1：Examination
Agent R2=10ul：200ul：50ul.Testing result refers to Fig. 1.
With reference to Fig. 1, it has been found that reagent of the present invention is after lasting 4 months, and under the conditions of 4 DEG C, reagent is almost without any
Attenuation, but contrast agents started to decay at the 30th day, had decayed nearly 50% after 4 months；Under the conditions of 37 DEG C, this hair
Bright reagent lasts 30 days, does not decay, but starts to decay thereafter, and attenuation 10% or so after 4 months, contrast agents are at the 9th day
When have begun to decay, after 4 months decay 40% or so；Under the conditions of 45 DEG C, reagent of the present invention starts to decay after 15 days, but
Be from 15 days to 120 day during almost no longer decay, contrast agents started to decay at the 6th day, after 120 days attenuation reach
50% or so.Therefore in the detection of total bilirubin heavy nitrogen, stability is greatly increased kit of the present invention, but
It is Shortcomings.
Anti-interference is tested
Lecithin is bought, the serum solution of 15g/L is prepared with pooled serum, is temporarily referred to as A liquid.Then it is prepared by doubling dilution
Into the quality-control product of different gradients, i.e. A liquid, 1/2A liquid, 1/4A liquid, 1/8A liquid.EDTA-2Na preparations are same as above, and A liquid is a concentration of
20g/L.It is tested with auspicious 800 automatic clinical chemistry analyzer advanced in years, using conventional heavy nitrogen total bilirubin kit conduct
Contrast agents, kit of the present invention are prepared using embodiment 2, and operating method no longer repeats, and testing result refers to figure
2。
Testing result according to fig. 2, it has been found that lecithin be for heavy nitrogen total bilirubin detection reagent box it is influential,
Kit of the present invention can resist the lecithin of 15g/L, but be below target value 10% or so, contrast agents are with lecithin lipid concentration
Increase, testing result gradually reduces, and when lecithin reaches 15g/L, has been higher by target value 200%；To being interfered in EDTA-2Na
For substance, kit of the present invention is almost unaffected, but with the raising of EDTA-2Na concentration, contrast agents detection knot
Fruit is stepped up, and when EDTA-2Na reaches 20g/L, testing result is higher by target value 200% or so, therefore interferes with substance lecithin
For fat and EDTA-2Na, the interference performance that both kit of the present invention is resisted is much better than contrast agents.
Clinical sample detection experiment
Kit of the present invention tests random N=40 sample with contrast agent box, and test result is in table form under
Table is embodied, the numerical value counted by the table 3, and then is calculated, the kit of kit of the present invention and market circulation
Correlation.Kit of the present invention is detected using the formula of embodiment 3.Testing result refers to Fig. 3.
Illustrate that kit of the present invention is when clinical sample detects, correlation r=0.9982 of the two with reference to Fig. 3 and Fig. 4.
In conclusion a kind of heavy nitrogen total bilirubin detection reagent box of stabilization of kit of the present invention is detected in clinical sample
When with contrast agents box have preferable correlation, it is but excellent in terms of reagent stability and to the antijamming capability of interfering substance
In contrast agents, good development space is provided for kit of the present invention, while enhance kit of the present invention on the market
Competitiveness.
Claims (3)
1. the heavy nitrogen total bilirubin detection reagent box stablized, it is characterised in that：The present invention is a kind of liquid-type double reagent detection
Kit, the kit include reagent R1 and R2；
The reagent R1 includes：
Disodium ethylene diamine tetraacetate 0.1%-0.5%
Aminobenzenesulfonic acid 50mmol/L-100mmol/L
Sodium chloride 5g/L-9g/L
D- glucopyranosides 0.05%-0.1%
D- glucopyranoses 0.075%-0.2%
Glucan 0.3-0.5%
Triton X-405 0.125%-0.4%
Citric acid-glycine buffer 0.1mol/L
The reagent R2 includes：
2,4 dichloro aniline diazol 50mmol/L-100mmol/L
Ethylenediamine tetra-acetic acid 0.1%-1%
Hydrochloric acid 0.1mol/L-0.4mol/L
Glycine-HCI buffer solution 0.1mol/L
D- glucopyranosides 0.05%-0.1%
D- glucopyranoses 0.075%-0.2%
Glucan 0.3%-0.5%
1-hydroxy ethylidene-1,1-diphosphonic acid 10mmol/L-20mmol/L.
2. kit according to claim 1, it is characterised in that：D- glucopyranosides in the reagent R1 and R2,
D- glucopyranoses, four kinds of substances of 1-hydroxy ethylidene-1,1-diphosphonic acid and glucan are stabilizer.
3. according to kit described in claim 1, it is characterised in that：Triton X-405 in the reagent R2 are that surface is lived
Property agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711427129.0A CN108181250A (en) | 2017-12-26 | 2017-12-26 | A kind of heavy nitrogen total bilirubin detection reagent box of stabilization |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711427129.0A CN108181250A (en) | 2017-12-26 | 2017-12-26 | A kind of heavy nitrogen total bilirubin detection reagent box of stabilization |
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| Publication Number | Publication Date |
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| CN108181250A true CN108181250A (en) | 2018-06-19 |
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ID=62547465
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|---|---|---|---|
| CN201711427129.0A Withdrawn CN108181250A (en) | 2017-12-26 | 2017-12-26 | A kind of heavy nitrogen total bilirubin detection reagent box of stabilization |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110261625A (en) * | 2019-07-15 | 2019-09-20 | 三诺生物传感股份有限公司 | A kind of bilirubin multiplexed detection reagents box and its application method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106053461A (en) * | 2016-08-18 | 2016-10-26 | 山东博科生物产业有限公司 | Stable diazo-method direct bilirubin detection kit |
-
2017
- 2017-12-26 CN CN201711427129.0A patent/CN108181250A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106053461A (en) * | 2016-08-18 | 2016-10-26 | 山东博科生物产业有限公司 | Stable diazo-method direct bilirubin detection kit |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110261625A (en) * | 2019-07-15 | 2019-09-20 | 三诺生物传感股份有限公司 | A kind of bilirubin multiplexed detection reagents box and its application method |
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Application publication date: 20180619 |