CN106501244A - Serum copper ions quantitative detecting reagent - Google Patents
Serum copper ions quantitative detecting reagent Download PDFInfo
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- CN106501244A CN106501244A CN201610872679.2A CN201610872679A CN106501244A CN 106501244 A CN106501244 A CN 106501244A CN 201610872679 A CN201610872679 A CN 201610872679A CN 106501244 A CN106501244 A CN 106501244A
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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
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
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Abstract
The invention discloses a kind of serum copper ions quantitative detecting reagent, including reagent R1, reagent R2 and calibration object, it is characterised in that：The reagent R1 includes：Buffer solution, interfacial agent, reaction promoter, stabilizer, bacteriostatic agent, wherein, the pH value of buffer solution is 2.0 5.0；The reagent R2 includes：Buffer solution, 3,5 DiBr PAESA, 0.01 0.1g/L, electrolyte, stabilizer, bacteriostatic agent, wherein, the pH value of buffer solution is 6.0 9.0；The calibration object includes：Buffer solution, copper ion, bacteriostatic agent, wherein, the pH value of buffer solution is 6.0 8.0.Advantage for present invention is：Relatively stable and preferable with atomic absorption method correlation.
Description
Technical field
The present invention relates to reagent technique field, more particularly, to a kind of serum copper ions quantitative detecting reagent.
Background technology
From nineteen twenty-eight it is found that copper be daily ration composition required composition after, up to nineteen fifty copper in Animal nutrition
Effect just little by little recognized.Copper is one of trace element necessary to livestock and poultry, and it is tyrosinase, monoamine oxidase, surpasses
The constituent of multiple copper enzymes such as superoxide dismutase, cytochrome oxidase, while also participate in cromoci, Vitamin C
Acid oxidase, the synthesis of galactase, thus with wide biological effect.Serum copper is copper in reflection livestock and poultry body
One important parameter of content, and carry out an important indicator of biochemistry test.
Copper is content occupies second in human body essential trace element, is one group in about 100 150mg of people's in-vivo content
The part of important enzyme, including CER (ferrous oxidase), superoxide dismutase, cytochrome oxidase, bad ammonia
Acid oxidase, dopamine β-hydroxylase and tyrosinase.
Serum copper increases and sees：Oral contraceptive, estrin treatment, Hodgkin's disease, leukaemia and other many malignant diseases
Change, megaloblastic anemia, alpastic anemia, hyperpigmentary disorders, rheumatic fever, heavy type and minor thalassemia, wound
And collagenosis.
Serum copper reduces seeing：Hepatolenticular degeneration (hepatolenticular degeneration), Menkes or silk volume syndrome, fire victim, certain
A little hypoferric anemia, protein malnutrition and chronic ischemic heart diseases etc..
Clinical assays serum copper method mainly has atomic absorption spectrophotometry and colorimetric method at present.Atomic absorption method spirit
Quick, special, but expensive atomic absorption spectrophotometer is needed, and cumbersome, time-consuming, clinical practice is less.Chemical method
Easy to operate, reagent is easy to get, and is easier to use in clinical expansion.
Content of the invention
It is an object of the invention to provide a kind of serum copper ions quantitative detecting reagent, it has relatively stable, and and atom
The characteristics of absorption process correlation is preferable.
The technical solution adopted in the present invention is：
Serum copper ions quantitative detecting reagent, including reagent R1, reagent R2 and calibration object, it is characterised in that：
The reagent R1 includes：Buffer solution 50-200mmol, interfacial agent 2.0-20.0g/L, reaction promoter 2.0-
12.0g/L, stabilizer 0.1-20.0g/L, bacteriostatic agent 0.1-10.0g/L, wherein, the pH value of buffer solution is 2.0-5.0；
The reagent R2 includes：Buffer solution 50-500mmol, 3,5-DiBr-PAESA 0.01-0.1g/L, electrolyte 100-
400mmol, stabilizer 0.1-20.0g/L, bacteriostatic agent 0.1-10.0g/L, wherein, the pH value of buffer solution is 6.0-9.0；
The calibration object includes：Buffer solution 20-300mmol, 20-60 μm of ol/L of copper ion, bacteriostatic agent 0.1-10.0g/L,
Wherein, the pH value of buffer solution is 6.0-8.0.
The buffer solution is Chinese holly edge acid buffer, acetate buffer, HEPES buffer solution, the one kind in PIPES buffer solutions.
The interfacial agent be Tween 20, Tween40, Span20, Span40, Span80, TritonX-100,
One kind in 20AB.
The reaction promoter is SDS, Brij 35, KAO 24B, one or more in methyl alcohol.
The electrolyte be sodium ion, potassium ion, magnesium ion, one or more in calcium ion.
The stabilizer be uric acid, lipoic acid, ascorbic acid, sodium ascorbate, one or more in hydroxylamine hydrochloride.
The bacteriostatic agent is methyl alcohol, potassium sorbate, Proclin200, Proclin300, the one kind in gentamicin.
Advantage for present invention is：
1st, detect quick.Can be grasped using automatic biochemistry analyzer using the serum copper ions quantitative detecting reagent of the present invention
Make, can be carried out with other detection projects simultaneously, each pattern detection time is shortened in 10min, is detected fast and convenient.
2nd, detection sensitivity ratio is high.The present invention serum copper ions quantitative detecting reagent by Reasonable adjustment reagent buffer,
The proportioning of the compositions such as interfacial agent, improves the sensitivity of reaction.
3rd, range of linearity width.The range of linearity of the serum copper ions quantitative detecting reagent of the present invention is 3-78.5 μm of ol/L,
Normal population term of reference is the male sex：11.0-22.0 μm ol/L, women：12.6-24.4 μm ol/L, children：12.6-29.9μ
Mol/L, disclosure satisfy that clinical needs.
4th, relatively stable.The each composition proportion of serum copper ions quantitative detecting reagent of the invention is more excellent, and reagent is close at 2-8 DEG C
Can stablize 12 months under the conditions of closing, after uncapping, store 2-8 DEG C, R1 stablizes 20 days, and R2 stablizes 20 days.
5th, correlation is preferable.Through actual verification, the serum copper ions quantitative detecting reagent and atomic absorption method of the present invention
Coefficient correlation be about 0.9658, illustrate that the reagent and atomic absorption method have preferably correlation.
Description of the drawings
The present invention is further described with reference to the accompanying drawings and examples：
Fig. 1 is that to carry out 40 parts of pattern detection results to contrast agents related for the serum copper ions quantitative detecting reagent of the present invention
Property test graph of a relation.
Specific embodiment
Embodiment 1
Serum copper ions quantitative detecting reagent, including reagent R1, reagent R2 and calibration object.
Specifically：
Reagent R1 includes：Buffer solution 150mmol, interfacial agent 5.5g/L, reaction promoter 8g/L, stabilizer 10g/L,
Bacteriostatic agent 0.8g/L.Wherein, buffer solution adopts pH value sour for 2.8 Chinese holly edge, and interfacial agent is using Triton X-100, reaction
Accelerator adopts SDS, stabilizer to adopt sodium ascorbate, bacteriostatic agent to adopt methyl alcohol.
Reagent R2 includes：Buffer solution 300mmol, 3,5-DiBr-PAESA 0.08g/L, electrolyte 150mmol, stabilizer
3g/L, bacteriostatic agent 0.9g/L.Wherein, buffer solution adopts pH value sour for 7.8 Chinese holly edge, and electrolyte adopts NaOH, stabilizer
Using ascorbic acid, bacteriostatic agent adopts methyl alcohol.
Calibration object includes：Buffer solution 30mmol, 50 μm of ol/L of copper ion, bacteriostatic agent 0.9g/L.Wherein, buffer solution adopts pH
It is worth the Chinese holly edge acid for 7.8, copper ion adopts methyl alcohol from copper sulphate, bacteriostatic agent.
Using method：
Instrument：7180 automatic biochemistry analyzer of Hitachi；
Assay method：2 points of end-point methods；
Detection wavelength：570nm；
The Direction of Reaction：Rise reaction；
V samples:VR1:VR2=15 μ l:240μl:60μl；
Calibration and calibrating mode：Calibrated using linear after water and calibration object calibration.
Operating procedure：
Sample and reagent R1 are mixed and are incubated 3-5min after 37 DEG C, read the 1st reading point absorbance A 1, add reagent immediately
R2, after mixing, 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2.
As a result calculate：
Calibrated with water (zero point) using calibration object, by 2 points of absorbance change values (A calibration-A are blank) to its phase
Answer concentration to do figure, draw calibration curve.(A samples-A is blank) value according to sample tries to achieve copper ions in sample on calibration curve
Content.
Theoretically, as reagent R1 provides the liquid environment of Copper in Serum ion and substrate reactions, with can make
The characteristics of copper ion becomes free state.Reagent R2 has the 3,5-DiBr-PAESA for making certain concentration in 2-8 DEG C of preservation condition
The characteristics of stably preserving down.Especially 3,5-DiBr-PAESA is only reacted with copper ion, is not reacted with other ions.The calibration object
Middle people's copper ion source is in copper sulphate or copper chloride.
When specifically used, the serum copper ions quantitative detecting reagent adopts colorimetric method, reaction principle be copper in sample from
After son is met in a liquid with reagent R1, dissociate with the material that script is combined, substrate (3,5-DiBr- corresponding to reagent
PAESA) meet in the liquid phase and react, make absorbance change.The absorbance change in the presence of enough substrates with
In sample, the concentration of copper ion is proportional, and absorbance change is compared with the calibration object of concentration known, can quantitatively draw sample
The content of middle copper ion.
Embodiment 2
Serum copper ions quantitative detecting reagent, including reagent R1, reagent R2 and calibration object.
Specifically：
Reagent R1 includes：Buffer solution 200mmol, interfacial agent 7.0g/L, reaction promoter 7g/L, stabilizer 12g/L,
Bacteriostatic agent 0.8g/L.Wherein, buffer solution adopt pH value for 3.6 acetic acid, interfacial agent adopts 20AB, and reaction promoter is adopted
SDS, stabilizer adopt ascorbic acid, bacteriostatic agent to adopt Proclin300.
Reagent R2 includes：Buffer solution 200mmol, 3,5-DiBr-PAESA 0.09g/L, electrolyte 150mmol, stabilizer
5g/L, bacteriostatic agent 0.9g/L.Wherein, buffer solution adopt pH value for 7.8 lemon acetic acid, electrolyte adopts NaOH, stabilizer
Using uric acid, bacteriostatic agent adopts methyl alcohol.
Calibration object includes：Buffer solution 30mmol, 50 μm of ol/L of copper ion, bacteriostatic agent 0.9g/L.Wherein, buffer solution adopts pH
It is worth the Chinese holly edge acid for 7.8, copper ion adopts Proclin200 from copper sulphate, bacteriostatic agent.
Using method, operating procedure, result calculate same as Example 1.
Embodiment 3
Serum copper ions quantitative detecting reagent, including reagent R1, reagent R2 and calibration object.
Specifically：
Reagent R1 includes：Buffer solution 50mmol, interfacial agent 2.0g/L, reaction promoter 2g/L, stabilizer 0.1g/L,
Bacteriostatic agent 0.1g/L.Wherein, buffer solution adopts pH value sour for 2 Chinese holly edge, and interfacial agent adopts Tween 20, reaction promoter
Using Brij 35, SDS, stabilizer adopts lipoic acid, bacteriostatic agent to adopt potassium sorbate.
Reagent R2 includes：Buffer solution 50mmol, 3,5-DiBr-PAESA 0.01g/L, electrolyte 100mmol, stabilizer
0.1g/L, bacteriostatic agent 0.1g/L.Wherein, buffer solution adopt pH value for 6.0 HEPES buffer solution, electrolyte adopts potassium hydroxide,
Stabilizer adopts hydroxylamine hydrochloride, bacteriostatic agent to adopt gentamicin.
Calibration object includes：Buffer solution 20mmol, 20 μm of ol/L of copper ion, bacteriostatic agent 0.1g/L.Wherein, buffer solution adopts pH
It is worth the PIPES for 6.0, copper ion adopts gentamicin from copper sulphate, bacteriostatic agent.
Using method, operating procedure, result calculate same as Example 1.
Embodiment 4
Serum copper ions quantitative detecting reagent, including reagent R1, reagent R2 and calibration object.
Specifically：
Reagent R1 includes：Buffer solution 160mmol, interfacial agent 20.0g/L, reaction promoter 12g/L, stabilizer 20g/
L, bacteriostatic agent 10g/L.Wherein, buffer solution adopt pH value for 5.0 acetic acid, using Tween 40, reaction promotes interfacial agent
Agent adopts KAO 24B, SDS, lipoic acid and uric acid of the stabilizer using arbitrary proportion combination, bacteriostatic agent to adopt potassium sorbate.
Reagent R2 includes：Buffer solution 500mmol, 3,5-DiBr-PAESA 0.1g/L, electrolyte 400mmol, stabilizer
20.0g/L, bacteriostatic agent 10g/L.Wherein, buffer solution adopt pH value for 9.0 HEPES buffer solution, electrolyte adopts magnesium hydroxide,
Stabilizer using arbitrary proportion combination uric acid, lipoic acid, ascorbic acid, sodium ascorbate, hydroxylamine hydrochloride, bacteriostatic agent adopt
Proclin300.
Calibration object includes：Buffer solution 300mmol, 60 μm of ol/L of copper ion, bacteriostatic agent 10g/L.Wherein, buffer solution adopts pH
It is worth the PIPES for 8.0, copper ion adopts gentamicin from copper sulphate, bacteriostatic agent.
Using method, operating procedure, result calculate same as Example 1.
Embodiment 5
With differing only in for embodiment 1：Interfacial agent adopts Span20, reaction promoter to combine using arbitrary proportion
SDS, Brij 35, KAO 24B, electrolyte adopts calcium hydroxide.
Embodiment 6
With differing only in for embodiment 2：Interfacial agent adopts Span40, reaction promoter to combine using arbitrary proportion
SDS, Brij 35, KAO 24B, methyl alcohol, electrolyte using arbitrary proportion combination NaOH, potassium hydroxide, hydroxide
Magnesium.
Embodiment 7
With differing only in for embodiment 3：Interfacial agent adopts Span80, reaction promoter to combine using arbitrary proportion
SDS, methyl alcohol, electrolyte using arbitrary proportion combination potassium hydroxide, calcium hydroxide, stabilizer using arbitrary proportion combination
Uric acid, lipoic acid, ascorbic acid, sodium ascorbate, hydroxylamine hydrochloride.
Effect example
1st, study on the stability：
The reagent of embodiment 1 is uncapped, after calibration, is placed in instrument reagent storehouse, determine same quality-control product daily, determine 24 days.
As a result, such as table 1, after showing that serum copper ions quantitative detecting reagent of the present invention is uncapped 20 days, measure Quality Control still, in the range of, meets
Clinical practice needs.
Table 1：Reagent of the present invention is uncapped estimation of stability
According to preceding method, identical experiment is carried out to the reagent of embodiment 2 to 7, as a result similar to table 1, illustrate to implement
The reagent of example 2 to 7 equally has preferably stability.
2nd, correlation is investigated
In this effect example, correlation analysis is to represent mutual linear relation between two variables with coefficient correlation (r), and sentences
The statistical method of its level of intimate of breaking.Correlation coefficient r does not have unit, changes in the range of -1+1, its absolute value closer to 1,
Linear correlation between two variables is closer, closer to 0, related not closer.If coefficient correlation just, illustrates a variable with another
One variable increases and decreases and increases and decreases, and direction is identical；If negative, represent that a variable increases, another variable is reduced, i.e., in opposite direction, but it
The relation of (such as various curves) beyond straight line can not being expressed.Correlation coefficient r=0 0.3 represent degree of correlation less than common, related
Coefficient r=0.3 0.5 represents that degree of correlation is common, and correlation coefficient r=0.5 0.7 represent degree of correlation significantly, correlation coefficient r
=0.7 0.9 represents that degree of correlation is high, and correlation coefficient r=0.9 1.0 represent that degree of correlation is high.
The reagent of Example 2, is compared test with atomic absorption method, contrast agents arrange parameter to specifications,
Respectively using each self-check system, to copper ion quantitative determination in identical 40 parts of serum samples.As a result as shown in Table 2 and Figure 1, relate to
And 40 samples, coefficient correlation is 0.9658, shows serum copper ions quantitative detecting reagent of the present invention compared with contrast agents,
There is good correlation.
Table 2：Reagent of the present invention and 40 parts of pattern detection results of contrast agents (atomic absorption method)
According to preceding method, identical experiment is carried out to the reagent of embodiment 1 and embodiment 3 to 7, as a result similar to table
2, illustrate that embodiment 1 and the reagent of embodiment 3 to 7 equally have good correlation.
The preferred embodiments of the present invention are the foregoing is only, the scope of the claims of the present invention, every utilization is not thereby limited
Equivalent structure or equivalent flow conversion that present specification and accompanying drawing are made, or it is related to be directly or indirectly used in other
Technical field, be included within the scope of the present invention.
Claims (7)
1. serum copper ions quantitative detecting reagent, including reagent R1, reagent R2 and calibration object, it is characterised in that：
The reagent R1 includes：Buffer solution 50-200mmol, interfacial agent 2.0-20.0g/L, reaction promoter 2.0-
12.0g/L, stabilizer 0.1-20.0g/L, bacteriostatic agent 0.1-10.0g/L, wherein, the pH value of buffer solution is 2.0-5.0；
The reagent R2 includes：Buffer solution 50-500mmol, 3,5-DiBr-PAESA 0.01-0.1g/L, electrolyte 100-
400mmol, stabilizer 0.1-20.0g/L, bacteriostatic agent 0.1-10.0g/L, wherein, the pH value of buffer solution is 6.0-9.0；
The calibration object includes：Buffer solution 20-300mmol, 20-60 μm of ol/L of copper ion, bacteriostatic agent 0.1-10.0g/L, wherein,
The pH value of buffer solution is 6.0-8.0.
2. serum copper ions quantitative detecting reagent according to claim 1, it is characterised in that：The buffer solution is the acid of Chinese holly edge
One kind in buffer solution, acetate buffer, HEPES buffer solution, PIPES buffer solutions.
3. serum copper ions quantitative detecting reagent according to claim 1, it is characterised in that：The interfacial agent is
One kind in Tween 20, Tween40, Span20, Span40, Span80, Triton X-100,20AB.
4. serum copper ions quantitative detecting reagent according to claim 1, it is characterised in that：The reaction promoter is
One or more in SDS, Brij 35, KAO 24B, methyl alcohol.
5. serum copper ions quantitative detecting reagent according to claim 1, it is characterised in that：The electrolyte be sodium from
One or more in son, potassium ion, magnesium ion, calcium ion.
6. serum copper ions quantitative detecting reagent according to claim 1, it is characterised in that：The stabilizer be uric acid,
One or more in lipoic acid, ascorbic acid, sodium ascorbate, hydroxylamine hydrochloride.
7. serum copper ions quantitative detecting reagent according to claim 1, it is characterised in that：The bacteriostatic agent be methyl alcohol,
One kind in potassium sorbate, Proclin200, Proclin300, gentamicin.
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| CN201610872679.2A CN106501244A (en) | 2016-09-30 | 2016-09-30 | Serum copper ions quantitative detecting reagent |
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|---|---|---|---|
| CN201610872679.2A CN106501244A (en) | 2016-09-30 | 2016-09-30 | Serum copper ions quantitative detecting reagent |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110596088A (en) * | 2019-09-20 | 2019-12-20 | 南京林业大学 | Method for rapidly determining copper ions |
| CN111141913A (en) * | 2020-01-02 | 2020-05-12 | 四川纳海川生物科技有限公司 | Ceruloplasmin detection kit and preparation method thereof |
| CN113720836A (en) * | 2021-09-17 | 2021-11-30 | 北京安图生物工程有限公司 | Kit for detecting serum copper ions and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110596088A (en) * | 2019-09-20 | 2019-12-20 | 南京林业大学 | Method for rapidly determining copper ions |
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| CN111141913A (en) * | 2020-01-02 | 2020-05-12 | 四川纳海川生物科技有限公司 | Ceruloplasmin detection kit and preparation method thereof |
| CN113720836A (en) * | 2021-09-17 | 2021-11-30 | 北京安图生物工程有限公司 | Kit for detecting serum copper ions and preparation method thereof |
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Application publication date: 20170315 |