CN108107199B - A kind of detection kit of luminol chemiluminescence analysis measurement uric acid - Google Patents
A kind of detection kit of luminol chemiluminescence analysis measurement uric acid Download PDFInfo
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- CN108107199B CN108107199B CN201711397762.XA CN201711397762A CN108107199B CN 108107199 B CN108107199 B CN 108107199B CN 201711397762 A CN201711397762 A CN 201711397762A CN 108107199 B CN108107199 B CN 108107199B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of detection kit of luminol chemiluminescence analysis measurement uric acid.The present invention is oxidized to allantoin and hydrogen peroxide using uric acid enzymatic uric acid, and hydrogen peroxide directly reacts the high-intensitive chemical signal to form a kind of duration with luminous substrate (without peroxidase).Compared with traditional enzymic colorimetric and chemoluminescence method, this method does not need addition catalase catalyzing hydrogen peroxide and directly shines with substrate reactions, can be used for full-automatic illumination instrument, is able to satisfy clinical automation mass detection.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of detection kit of luminol chemiluminescence analysis measurement uric acid.
Background technique
Uric acid (2,4,6- trihydroxypurine, abbreviation UA) molecular formula is C5H4N4O3, molecular weight MW=168.11.It is
A kind of white crystalline substance, it is more difficult to it is dissolved in water, is insoluble in acid, be also insoluble in ether and ethyl alcohol, there is alkalescent, it can be with strong acid
At salt.Uric acid exists in a salt form in vivo, thus solubility is higher.Uric acid be in human body purine metabolism produce eventually
Object, it is most of to be excreted with urine, it is discharged at least partially through excrement and sweat.Plasma Uric Acid normal value: male: 149~416 μ
Mol/L, female: 89~357 μm of ol/L.It is known as hyperuricemia when uric acid concentration is excessively high in purine metabolic disturbance in human body, blood plasma
(HUA).The solubility of uric acid is low, deposits in vascular wall and easily induces gout, secondary hypertension, cerebral infarction, diabetes, congested
Heart failure, ureteral calculi and kidney trouble etc. cause renal failure or even threat to life when serious.
Uric acid is all from glomerular filtration in blood, wherein 98% in proximal tubular initial part reabsorption, then 50%
It is intracavitary that renal tubule is secreted into the middle part of proximal tubular again, is had again in proximal tubular end portion 40%~44% by reabsorption,
There is 6%~10% uric acid discharge.The generation of uric acid and excretion are more constant in normal human, and uric acid content changes in body fluid, can
Sufficiently to reflect the situation of the functions such as human body metabolism, immune.Therefore clinical detection serum uric acid concentration is to laboratory diagnosis pain
The diseases such as wind and kidney have important reference value.
There is a kind of chemical luminescence detection method in laboratory testing uric acid at present, such as Chinese patent application CN1912599
The chemical luminescent detecting method of disclosed a kind of uric acid in serum, characterized in that uric acid is oxidized under uric acid enzymatic, is generated
H2O2, under hydrogen peroxide enzyme effect, make H2O2Water and single oxygen are generated, single oxygen can make luminous substrate (generally luminol) oxygen
Change, issues optical signal, the size of optical signal and the concentration of serum uric acid are positively correlated.Serum uric acid with known concentration with measure
Luminous signal, dose-response curve is made, to go out the uric acid content in sample to be tested according to the curve calculating.But it should
Method is need to generally to add the catalyst such as peroxidase using luminol, different luminol and its derivative as luminous substrate,
The stability of these reagent components is poor, and the specificity of peroxidase is not high, since hydrogen peroxide is in the reaction
As main intermediate product, thus it is easy to be interfered in continuous mode.
Secondly, what cannot most be ignored during detecting uric acid at present is exactly that endogenous material present in sample is for example blood red
Albumen, bilirubin and the exogenous material from clinical medicine application lead to institute if ascorbic acid is interfered caused by reaction
Result is surveyed to have a certain difference with true value.
Therefore, in view of the above technical problems, it is necessary to provide a kind of quality stabilization, and can be effectively anti-interference, accuracy is high
For detecting the kit of uric acid in blood.
Summary of the invention
The present invention is intended to provide a kind of detection kit of luminol chemiluminescence analysis measurement uric acid, the kit utilize uricase
Catalysis uric acid is oxidized to allantoin and hydrogen peroxide, and hydrogen peroxide directly reacts to form a kind of the high-strength of duration with luminous substrate
Chemical signal (being not necessarily to peroxidase) is spent, determinand content is calculated by the optical signal that hydrogen peroxide and substrate generate, meanwhile,
Its strong antijamming capability, accuracy is high, is more suitable for clinical use.
In order to achieve the above object, the invention adopts the following technical scheme: a kind of inspection of luminol chemiluminescence analysis measurement uric acid
Test agent box, including reagent R1, reagent R2 and reagent R3, the reagent R1 include following components and concentration:
The reagent R2 includes following components and concentration:
The component of the reagent R3 are as follows:
Luminous substrate: Lumigen HyPerBlu.
Further, in the reagent R1 pH of buffer be 6.5-8.5, selected from phosphate buffer, Tris buffer,
One or more of acetate buffer solution and HEPES buffer solution.
Further, pH of buffer is 6.5-8.5 in the reagent R1, is phosphate buffer.
Further, the reagent R1 anti-interference agent I is selected from ascorbic acid oxidase, potassium ferrocyanide, the potassium ferricyanide, gallbladder
One or more of red pigment oxidizing ferment and catalase.
Further, the reagent R1 anti-interference agent I is ascorbic acid oxidase and bilirubin oxidase.
Further, stabilizer described in the reagent R1 and R2 is selected from bovine serum albumin(BSA), trehalose, sucrose, sweet dew
One or more of alcohol, disodium ethylene diamine tetraacetate and glycerol.
Further, stabilizer described in the reagent R1 and R2 is bovine serum albumin(BSA) and trehalose.
Further, the pH of buffer in the reagent R2 is 8.0-10, is selected from borate buffer solution, glycine-hydrogen
One or more of sodium oxide molybdena buffer, Tris buffer and phosphate buffer.
Further, the pH of buffer in the reagent R2 is 8.0-10, is borate buffer solution.
Further, reinforcing agent described in the reagent R2 is Tween 80 primary degradation object.
Further, the Tween 80 primary degradation object is made by following steps:
Tween 80 solution, the green wood that inoculation 1~3wt% concentration is 800~1000cfu/mL are added in 100ml shaking flask
Mould, setting temperature is 30~40 DEG C, revolving speed 120r/min, cultivates 8~12h, and centrifugation takes supernatant；Successively supernatant is carried out micro-
Filter, cation exchange and sterilizing, are made Tween 80 primary degradation object.
Further, preservative described in the reagent R1 and R2 is selected from Sodium azide, proclin 300 and KY100 biology
One or more of preservative.
Further, preservative described in the reagent R1 is KY100 biological preservative, and preservative described in reagent R2 is nitrine
Sodium.
The present invention provides a kind of uric acid detection kit that difference is previous, testing principles are as follows: uric acid is in uricase
Under effect, hydrogen peroxide is generated, hydrogen peroxide directly reacts to form a kind of continual and steady chemical signal with luminous substrate, pass through
Luminous intensity is detected, is compared with standard curve, the content of uric acid in sample can be extrapolated.The present invention can obtain said effect
Key point is, which can directly and hydroperoxidation can shine under alkaline environment.Itself and previous enzyme ratio
Color method is compared with chemoluminescence method, and most important difference is, is free of peroxidase, i.e., there is no hydrogen peroxide oxidation is anti-
It answers, hydrogen peroxide non-dependent enzymatic not as intermediate product, the directly concentration of detection hydrogen peroxide in detection reaction process
Reaction, thus the interference being subject in detection process can be greatly reduced.
Another of the invention key point is, the present invention is by being added ascorbic acid oxidase in reagent R1 and gallbladder is red
Plain oxidizing ferment is added Tween 80 primary degradation object as reinforcing agent in reagent R2, can significantly increase as anti-interference reagent I
The anti-interference ability of strong kit, to be effectively removed Ascorbic Acid in Blood Serum, bilirubin, pyruvic acid, triglycerides and blood
The interference of Lactoferrin substance improves the accuracy of detection kit, higher than conventional enzyme process sensitivity and accuracy.
It is emphasized that the initial thinking of inventor is using Tween 80 as reinforcing agent, but due to reagent of the present invention
R1 and R2 is alkaline solution, and Tween 80 is easy hydrolysis under alkaline condition, is theoretically using Tween 80 as reinforcing agent as a result,
It is infeasible.And inventor is inoculated with Trichoderma viride culture it was unexpectedly observed that with Tween 80 for unique carbon source, harvests supernatant system
The Tween 80 primary degradation object obtained can obtain preferable synergy with anti-interference reagent I as reinforcing agent.Also, it invents
People also found, joined Tween 80 primary degradation object, and the BSA as stabilizer can also be overcome to be easy not under alkaline environment
Stable problem has stabilization to a certain extent to BSA.
The invention has the following advantages that
1) the present invention provides a kind of kit for detecting uric acid in blood content, peroxidase is not contained, is disobeyed
Rely enzymatic reaction, but by the concentration of detection peroxide, therefore the interference being subject in detection process can be greatly reduced, and be made
It obtains of the invention with higher sensitivity, accuracy and better repeatability.
2) ascorbic acid oxidase and bilirubin oxidase is added as anti-interference reagent I in the present invention in reagent R1,
Tween 80 primary degradation object is added in reagent R2 as reinforcing agent, kit can be significantly increased to the anti-interference energy of chaff interferent
Power, so that it is effectively removed the interference of Ascorbic Acid in Blood Serum, bilirubin, pyruvic acid, triglycerides and hemoglobin material, and
The influence of chromogen in sample or reagent is not will receive, so that result is more accurate.
Detailed description of the invention
Fig. 1 is canonical plotting, and wherein X-axis indicates that the log value of UV concentration, Y-axis indicate the log value of luminous intensity；
Fig. 2 is the correlation comparison diagram of 2 reagent of the embodiment of the present invention and the testing uric acid kit of Beijing the last nine, wherein X
Axis indicate 2 kit measurement of the embodiment of the present invention serum as a result, Y-axis indicate be Beijing the last nine kit measurement serum knot
Fruit.
Specific embodiment
The specific embodiment of form by the following examples makees further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following embodiment.
Luminous substrate Lumigen HyPerBlu of the present invention is purchased from U.S. Lumigen Inc.
The preparation of embodiment 1, Tween 80 primary degradation object
Tween 80 solution is added in 100ml shaking flask, is inoculated with the Trichoderma viride that 1~3wt% concentration is 1000cfu/mL, if
Setting temperature is 35 DEG C, revolving speed 120r/min, cultivates 12h, and centrifugation takes supernatant；Successively to supernatant carry out micro-filtration, 732 type sun from
Sub-exchange resin carries out cationic exchange and sterilizing, and Tween 80 primary degradation object is made.
The detection kit of embodiment 2, a kind of luminol chemiluminescence analysis measurement uric acid
Reagent R1:
Reagent R2:
Reagent R3:
Lumigen HyPerBlu。
The detection kit of embodiment 3, a kind of luminol chemiluminescence analysis measurement uric acid
Embodiment 3 the difference from example 2 is that, anti-interference agent I is ascorbic acid oxidase and ferrous cyanogen in reagent R1
Change potassium, the concentration of Tween 80 primary degradation object is 0.03w/v%, remaining parameter and operation are as described in Example 2.
The detection kit of embodiment 4, a kind of luminol chemiluminescence analysis measurement uric acid
Embodiment 4 the difference from example 2 is that, in reagent R1 anti-interference agent I be bilirubin oxidase and ferrocyanide
Potassium, the concentration of Tween 80 primary degradation object are 1w/v%, remaining parameter and operation are as described in Example 2.
The detection kit of embodiment 5, a kind of luminol chemiluminescence analysis measurement uric acid
Embodiment 5 is that buffer is Tris buffer in the reagent R1, and buffer is in R2 with the difference of embodiment 2
Glycine-NaOH buffer, remaining parameter and operation are as described in Example 2.
The detection kit of comparative example 1, a kind of luminol chemiluminescence analysis measurement uric acid
Comparative example 1 is with the difference of embodiment 2, does not contain reinforcing agent, remaining parameter and operation are as described in Example 2.
The detection kit of comparative example 2, a kind of luminol chemiluminescence analysis measurement uric acid
Comparative example 2 is that reinforcing agent is added in reagent R1 with the difference of embodiment 2, remaining parameter and operation are such as 2 institute of embodiment
Show.
Embodiment 6, detection method
Uric acid detection kit described in the embodiment of the present invention 2~5 is suitable for a plurality of types of full-automatic or semi-automatic hair
Light analyzer, by taking Coase steps SMART1000 full-automatic illumination instrument as an example, parameter such as table 1.
Analysis method: some end-point methods are added 90 μ L reagent R1 and 5 μ L samples in 37 DEG C of incubation 5min, 90 μ L are added
30 μ L luminous substrate R3 are added in reagent R2,37 DEG C of incubation 5min, detect luminous value RLU after reacting 5min, are calculated by instrument
To final detection result.Using the log value of the UA concentration of standard items as X-coordinate after measurement, the log value of luminous intensity RLU is as Y
Coordinate makes standard curve as shown in Figure 1, equation Y=1.0002X-0.3015, R2=1, sample is calculated according to the standard curve
The concentration of uric acid in product.
Table 1
Test example one, correlation test
Using the testing uric acid kit of reagent of the present invention (specific formula is with embodiment 2) and Beijing the last nine, it is respectively adopted
Coase steps SMART1000 full-automatic illumination instrument, Ku Beier iMagic-M7 automatic clinical chemistry analyzer presses 40 parts of fresh human serums
Respective parameter is measured simultaneously, carries out correlation regression analysis to measured value, measurement result is shown in Fig. 2.
Found out by the result of Fig. 2, the coefficient R of two kinds of reagents2=0.9996, regression equation y=1.0046x-
1.7609.The result shows that this reagent with listed contrast agent measurement patients serum's correlation it is good, have well specificity
And accuracy.
Test example two, accuracy and precision test
Reagent: reagent (specific formula is with embodiment 2) of the present invention, standard items, quality-control product.
Instrument: Coase steps SMART1000 full-automatic illumination instrument.
Operating procedure: being calibrated using standard items, measures each Quality Control 10 times, calculates test mean value, SD, CV and relative deviation.
For table 2 the results show that the relative deviation that reagent of the present invention detects each Quality Control is respectively less than 3%, accuracy is very good.Measurement
10 CV values with sample are respectively less than 2%, and precision is preferable.
2 test result of table (μm ol/L)
Test example three, linear test
Using reagent of the present invention (specific formula is with embodiment 2), standard items, high concentration uric acid sample, quality-control product.
Instrument: Coase steps SMART1000 full-automatic illumination instrument.
Operating procedure: using blank solution as dilution, by 5:1,5:2,5:3,5:4,5:5 (former times) dilution, each sample
Replication 3 times.
3 result of table indicates that reagent of the present invention is linear good within the scope of 0~2000 μm of ol/L, and the range of linearity is very wide.
3 test result of table (μm ol/L)
Test example four, anti-interference test
Fresh mix serum is taken, 5 equal portions are divided into, every equal portions are then separated into 10 equal portions, is added not according to the following table 4 concentration
Same interfering substance, takes 1~2 kit of the embodiment of the present invention 2~4 and comparative example, measures the content of uric acid in serum respectively,
Measurement result is as shown in table 5.
Relative deviation (%)=(measurement mean value-noiseless object sample measurement mean value of interference sample)/empty noiseless object
Measurement mean value × 100% of sample.
4 interfering substance concentration of table
5 testing result of table
As shown in Table 5, the kit strong antijamming capability of the embodiment of the present invention 2~4, can be substantially reduced Vitamin C
The influence that acid, bilirubin, pyruvic acid, triglycerides and hemoglobin detect kit has excellent anti-interference ability.And
Compared with Example 2, the anti-interference ability of 1 kit of comparative example (eliminating reinforcing agent), Ascorbic Acid and bilirubin without
It is decreased obviously, but the anti-interference ability of pyruvic acid, triglycerides and hemoglobin chaff interferent is remarkably decreased, testing result table
Bright existing negative interference is bigger；The anti-interference energy for the kit being prepared is added in reinforcing agent in R1 in 2 kit of comparative example
Power also has compared with Example 2 significantly to be declined.This explanation, compared in the prior art, kit of the present invention has more excellent
Anti-interference ability, thus have higher accuracy and sensitivity.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (8)
1. a kind of detection kit of luminol chemiluminescence analysis measurement uric acid, which is characterized in that including reagent R1, reagent R2 and reagent
R3, the reagent R1 include following components and its concentration:
The reagent R2 includes following components and concentration:
The component of the reagent R3 are as follows:
Luminous substrate: Lumigen HyPerBlu；Wherein, reinforcing agent described in the reagent R2 is Tween 80 primary degradation object；
The Tween 80 primary degradation object is made by following steps:
Tween 80 solution is added in 100ml shaking flask, is inoculated with the Trichoderma viride that 1~3wt% concentration is 800~1000cfu/mL,
It is 30~40 DEG C, revolving speed 120r/min that temperature, which is arranged, cultivates 8~12h, and centrifugation takes supernatant；Successively to supernatant carry out micro-filtration,
Cation exchange and sterilizing, are made Tween 80 primary degradation object.
2. detection kit as described in claim 1, which is characterized in that the pH of buffer is 6.5-8.5 in the reagent R1,
It is selected from one or more of phosphate buffer, Tris buffer, acetate buffer solution and HEPES buffer solution.
3. detection kit as described in claim 1, which is characterized in that anti-interference agent I described in the reagent R1 is selected from anti-
One or more of bad hematic acid oxidizing ferment, potassium ferrocyanide, the potassium ferricyanide, bilirubin oxidase and catalase.
4. detection kit as claimed in claim 3, which is characterized in that anti-interference agent I described in the reagent R1 is anti-bad
Hematic acid oxidizing ferment and bilirubin oxidase.
5. detection kit as described in claim 1, which is characterized in that stabilizer described in the reagent R1 and R2 is selected from ox
One or more of seralbumin, trehalose, sucrose, mannitol, disodium ethylene diamine tetraacetate and glycerol.
6. detection kit as claimed in claim 5, which is characterized in that stabilizer described in the reagent R1 and R2 is ox blood
Pure albumen and trehalose.
7. detection kit as described in claim 1, which is characterized in that the pH of buffer in the reagent R2 is 8.0-10,
It is selected from one of borate buffer solution, Glycine-NaOH buffer, Tris buffer and phosphate buffer or several
Kind.
8. detection kit as described in claim 1, which is characterized in that preservative described in the reagent R1 and R2 is selected from folded
One or more of nitrogen sodium, proclin 300 and KY100 biological preservative.
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| CN108982617A (en) * | 2018-08-01 | 2018-12-11 | 湖南海源医疗科技股份有限公司 | A kind of uric acid electrochemical test strip and preparation method thereof |
| CN110297082A (en) * | 2019-06-12 | 2019-10-01 | 杭州启创生物技术有限公司 | The method of the markers such as saliva uric acid specific detection and diagnosis gout |
Citations (4)
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|---|---|---|---|---|
| US4230798A (en) * | 1978-02-13 | 1980-10-28 | Miles Laboratories, Inc. | Unitized uric acid test composition and device |
| CN103571916A (en) * | 2013-11-22 | 2014-02-12 | 重庆医科大学 | Formula of kit for testing content of uric acid through double reagent method |
| CN103857393A (en) * | 2011-03-25 | 2014-06-11 | 葛兰素史密斯克莱知识产权（第2号）有限公司 | Cyclopropylamines as LSD1 inhibitors |
| CN106290323A (en) * | 2015-06-04 | 2017-01-04 | 章丘美高义医疗器械有限公司 | A kind of stable, uric acid reagent that capacity of resisting disturbance is strong and detection method |
-
2017
- 2017-12-21 CN CN201711397762.XA patent/CN108107199B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4230798A (en) * | 1978-02-13 | 1980-10-28 | Miles Laboratories, Inc. | Unitized uric acid test composition and device |
| CN103857393A (en) * | 2011-03-25 | 2014-06-11 | 葛兰素史密斯克莱知识产权（第2号）有限公司 | Cyclopropylamines as LSD1 inhibitors |
| CN103571916A (en) * | 2013-11-22 | 2014-02-12 | 重庆医科大学 | Formula of kit for testing content of uric acid through double reagent method |
| CN106290323A (en) * | 2015-06-04 | 2017-01-04 | 章丘美高义医疗器械有限公司 | A kind of stable, uric acid reagent that capacity of resisting disturbance is strong and detection method |
Non-Patent Citations (1)
| Title |
|---|
| 血清尿酸测定中维生素C氧化酶抗干扰能力研究;陈远翔等;《国际检验医学杂志》;20140130;第35卷(第2期);第208-212页 |
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Denomination of invention: A kit for determination of uric acid by enzyme chemiluminescence Effective date of registration: 20200910 Granted publication date: 20190212 Pledgee: Bank of China Limited by Share Ltd. Guangzhou Tianhe branch Pledgor: GUANGZHOU JINDE BIOTECHNOLOGY Co.,Ltd. Registration number: Y2020440000262 |
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