CN106404686A - Antiheparin serum total bilirubin (vanadate oxidation method) detection kit - Google Patents
Antiheparin serum total bilirubin (vanadate oxidation method) detection kit Download PDFInfo
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- CN106404686A CN106404686A CN201610735492.8A CN201610735492A CN106404686A CN 106404686 A CN106404686 A CN 106404686A CN 201610735492 A CN201610735492 A CN 201610735492A CN 106404686 A CN106404686 A CN 106404686A
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- total bilirubin
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- G—PHYSICS
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- 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
- 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
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/728—Bilirubin; including biliverdin
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Abstract
The invention belongs to the technical field of serum total bilirubin (vanadate oxidation method) detection, and especially relates to a serum total bilirubin detection reagent. The serum total bilirubin detection reagent comprises a reagent R1 and a reagent R2; the reagent R1 contains a disodium hydrogen phosphate-citric acid buffer solution, hexadecyl trimethyl ammonium bromide, fatty alcohol-polyoxyethylene ether, alkylphenol ethoxylates (OP), polyethylene glycol 8000, and lauryl sodium sulfate; and the reagent R2 contains a phosphate buffer solution, ethylenediamine tetraacetic acid disodium salt, and sodium metavanadate. The serum total bilirubin detection reagent is suitable to be used together with a plurality of fully automatic biochemical analyzers, and detection results are not influenced by heparin.
Description
Technical field
The present invention relates to serum total bilirubin (Vanadic acid oxidation) detection technique field, it is suitable for entirely certainly particularly to a kind of
Serum total bilirubin (Vanadic acid oxidation) detectable of Automatic Biochemical Analyzer.
Background technology
Total bilirubin isBilirubin directSummation with both unconjugated bilirubins.Total bilirubin source mainly has：80% 1.～
85% bilirubin is derived from old and feeble erythrocyte disintegrate;2. about 15% by erythrocyte quilt in bone marrow still immature in hematopoiesis
Destroy（Ineffectivity erythropoiesis in bone marrow）And formed;3. it is derived from heme-containing protein on a small quantity（hemoprotein）, such as flesh
The destruction of Lactoferrin, peroxidase, cytochrome etc. is decomposed.
Total bilirubin is mainly used to whether diagnosis has whether hepatic disease or biliary tract occur exception, and total bilirubin normal value exists
Between 3.4～17.1 μm of ol/L, 17.1-34.2 μm of ol/L can be considered recessive subcutaneous ulcer;34.2-170 being slight jaundice between μm ol/L;
170-340 μm of ol/L is moderate jaundice;It is then severe jaundice more than 340 μm of ol/L.
The serum total bilirubin detection method clinically commonly used has heavy nitrogen, Bilirubin Oxidase method and vanadate oxidation
Method.Heavy nitrogen reagent has higher experiment sensitivity, reagent low price, detection operational approach simple, but repeatability is unstable;
Bilirubin Oxidase method has that specificity is good, sensitivity is high, reproducible, the advantages of easy and simple to handle, but due to reagent price relatively
Height, widely uses at present not yet.Later with vanadate method detect blood mesobilirubin method appearance and at home by
Gradually promote, the evaluation in clinic is also more, the good specificity of its owner and stability have obtained the good of Clinical practice person
Comment, but the gradually popularization being as Clinical practice finds, processes, using heparin, the plasma sample obtaining in being directed to clinic,
During using vanadate method detection total bilirubin, cause testing result inaccurate, response curve exception and testing result is abnormal, hold
It is easily caused testing result low or even negative value occurs, this gives popularization in clinic and using causing very big puzzlement.
Content of the invention
It is directed to serum total bilirubin detection, the invention provides a kind of serum total bilirubin of antiheparin (vanadate method)
Detection kit, compared with common detection methods, testing result is not affected this test kit by heparin, and using simple, convenient, tool
There are good accuracy, repeated and good detection range, be conducive to reagent popularization and application clinically.
The present invention is achieved by the following measures：
Be used new surfactant replace original cationic surfactant cetyl trimethylammonium bromide as plus
Fast agent, destroys the total hydrogen bond of unconjugated bilirubin, is then fully exposed the bilirubin after accelerating the failure, attached in PH=3
Closely, vanadate is acted on sample, the total bilirubin in sample is oxidized to biliverdin, yellow specific to bilirubin reduces,
Detect that it is aoxidized the difference of before and after's absorbance, the content of the TBIL in its intensity of variation and sample by vanadate at wavelength 450nm
It is directly proportional.
A kind of total bilirubin detection reagent, including reagent R1, R2, the composition of described reagent R1, R2 is as follows：
Reagent R1：
Disodium hydrogen phosphate-benzoic acid buffer (pH2.9) 0.1mol/L
Fatty alcohol-polyoxyethylene ether 5g/L-10 g/L
Alkylphenol polyoxyethylene (OP) 4 g/L -8g/L
Macrogol 8000 1%-5.0%
TritonX -305 2.5 g/L -5g/L
Sodium fluoride 1.0g/L-2.0 g/L
Tetrasodium ethylenediamine tetraacetate 1g/L-2 g/L
Reagent R2:
Phosphate buffer (pH7.0) 10mmol/L
Disodiumedetate 5g/L-10g/L
Tetrasodium ethylenediamine tetraacetate 1g/L-2g/L
Sodium metavanadate 4mmol/L
Described reagent R1 when using：R2= 4:1, R1 consumption is 240 μ l.
The invention has the beneficial effects as follows：
1st, the limited benzoic acid that have selected of the present invention, as main buffer, both can promote vanadate that bilirubinic oxidation is made
With reaction system can be played with antiseptical effect again；
2nd, the nonionics such as fatty alcohol-polyoxyethylene ether, alkylphenol polyoxyethylene (OP), Polyethylene Glycol have been selected in the present invention
Surface activity is as bilirubinic accelerator, thus avoiding being polymerized of cationic surfactant and macromole heparin material,
And can be good at playing the effect of abundant acceleration, three kinds of surfactants have synergism；
3rd, in reagent while adding disodiumedetate, with the addition of a small amount of tetrasodium ethylenediamine tetraacetate chelating
Agent, can play a role to different heavy metal ion, and tetrasodium ethylenediamine tetraacetate can suppress vanadate to other things
The oxidation of matter, thus improve the specificity of testing result.
4th, this law is bright with the addition of TritonX -305 surfactant in reagent 1, it is possible to increase the clarification journey of reaction system
Degree, improves the precision of product.
5th, in the present invention, preferred sodium fluoride, as ionic equilibrium agent, adjusts course of reaction intermediate ion concentration to reaction
Impact.
Brief description
Fig. 1 is the correlation curve figure of two kinds of reagent；
Fig. 2 is reagent stability of the present invention;
Fig. 3 embodiment 1 reagent detects operational approach
Fig. 4 embodiment 2 reagent interference free performance compares；
Fig. 5 embodiment 1 reagent and market is common and the serum total bilirubin determination test kit comparison and detection result that gets the nod.
Specific embodiment
With reference to specific embodiment, the present invention is further described：
Embodiment 1
Disodium hydrogen phosphate-benzoic acid buffer (pH2.9) 0.1mol/L
Fatty alcohol-polyoxyethylene ether 5g/L-10 g/L
Alkylphenol polyoxyethylene (OP) 4 g/L -8g/L
Macrogol 8000 1%-5.0%
TritonX -305 2.5 g/L -5g/L
Sodium fluoride 1.0g/L-2.0 g/L
Tetrasodium ethylenediamine tetraacetate 1g/L-2 g/L
Reagent R2:
Phosphate buffer (pH7.0) 10mmol/L
Disodiumedetate 5g/L-10g/L
Tetrasodium ethylenediamine tetraacetate 1g/L-2g/L
Sodium metavanadate 4mmol/L
The using method of the present embodiment reagent：
Serum total bilirubin (Vanadic acid oxidation) detectable of the present embodiment description, is divided using full-automatic biochemical when using
Analyzer, such as Hitachi 7180 fully-automatic analyzer etc., it is measured using Two point end assay.The ratio of sample and reagent is set as
9：240：60, Detection wavelength is 450nm, places distilled water, standard substance and sample in the correspondence position of specimen disc, operation is as schemed
3.
Embodiment 2
Interference is tested：Take fresh mix serum, be divided into 2 equal portions, then every equal portions are separated into 5 equal portions, add different doing
Disturb material so as to the concentration in serum reaches the requirement of Fig. 4.Then respectively use embodiment 1 gained reagent, common with market simultaneously
The content of the serum total cholesterol reagent comparative determination serum total cholesterol simultaneously of accreditation, matched group measurement result is different with addition
After interfering material, the measurement result of each group is shown in Fig. 4.Relative deviation (%)=(mensure of the mensure average-check sample of interference sample
Average)/check sample mensure average × 100%.
As seen from Figure 4, ascorbic acid≤20mmol/L, heparin≤0.5%, hemoglobin≤200mg/L, glycerol three
Ester≤22.6mmol/L situation does not substantially interfere with to embodiment 1 reagent test result.Compared with results of comparison, reagent of the present invention
Antiheparin ability is substantially, consistent with reference product in the detection of other interfering materials, illustrate the reagent of embodiment 1 accuracy with
Up to standard on capacity of resisting disturbance.
Embodiment 3
Dependency is tested：Using embodiment 1 formula reagent preparation, the State Food and Drug Administration common with market is approved
Serum total bilirubin (Vanadic acid oxidation) test kit of certain company carry out control test, have detected 20 clinical serum simultaneously
Sample, testing result is as shown in Figure 5.And obtain the correlation curve of two kinds of reagent（As shown in Figure 1）, shown by testing result
Show, the correlation coefficient of two test kits is 0.999, illustrates that both have great dependency.
Calibration object used by test and quality-control product are respectively：
Calibration object：The content of RANDOX926 serum total bilirubin is 32.2 μm of ol/L.
Quality-control product：The target value of RANDOX1005 serum total cholesterol is 29.3 μm of ol/L, target value scope：23.2~35.4μ
mol/L.
Embodiment 4
Reagent stability checking test：
Using gained detectable in the embodiment of the present invention 1 ~ 3 as test group, take a kind of commercially available serum total bilirubin (vanadate oxygen
Change method) detection kit as a control group, test group with compare every group of group reagent and ask for two parts of identical, portion does 15 days and opens
Bottle stability test, reagent is placed in 2-8 DEG C of cold closet of instrument（Do not take out within 15 days）, stable as corkage in 15 days
Property detection；Another does 37 DEG C of heat stability test tests, and closing is placed in 37 DEG C of thermostat water baths（Daily only in detection
When take out, detection finish after, still sealing put back in 37 DEG C of water-baths, continuous 7 days）, test as 7 days 37 DEG C of heat stability
Card.By reagent simultaneously on Hitachi 7180 automatic clinical chemistry analyzer device, detected according to following Fig. 3 method, and in instrument
Upper Criterion curve.Take lyophilized powder quality-control product, after being uniformly dissolved, be divided into 15 parts, -20 DEG C of storages, daily Quality Control one,
And tracing detection result, it opens tracking and monitoring trend such as Fig. 2.
Claims (4)
1. a kind of serum total bilirubin of antiheparin (Vanadic acid oxidation) detection kit is it is characterised in that by reagent R1, R2
Composition, wherein R1 comprises disodium hydrogen phosphate-benzoic acid buffer (pH2.9), fatty alcohol-polyoxyethylene ether, alkylphenol-polyethenoxy
Ether (OP), Macrogol 8000, TritonX -305, sodium fluoride, tetrasodium ethylenediamine tetraacetate；R2 comprises phosphate buffer
(pH7.0), disodiumedetate, tetrasodium ethylenediamine tetraacetate, sodium metavanadate.
2. test kit according to claim 1 it is characterised in that described reagent use when R1：R2= 4:1, R1 consumption
For 240 μ l.
3. the present invention uses fatty alcohol-polyoxyethylene ether, alkylphenol polyoxyethylene (OP), poly- second two according to claim 1
The nonionic surfactant such as alcohol, it is to avoid reaction with heparin, make testing result not affected by heparin.
4. according to claim 1 in reagent add disodiumedetate while, with the addition of a small amount of second two
Amine tetraacethyl four sodium chelating agen, improves the specificity of testing result.
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| CN201610735492.8A CN106404686B (en) | 2016-08-27 | 2016-08-27 | A kind of serum total bilirubin of antiheparin (Vanadic acid oxidation) detection kit |
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| CN201610735492.8A CN106404686B (en) | 2016-08-27 | 2016-08-27 | A kind of serum total bilirubin of antiheparin (Vanadic acid oxidation) detection kit |
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| CN106404686B CN106404686B (en) | 2019-03-29 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109813918A (en) * | 2019-01-11 | 2019-05-28 | 河北省医疗器械与药品包装材料检验研究院(河北省医疗器械技术审评中心) | A kind of total bilirubin determination reagent kit |
| CN111077322A (en) * | 2019-12-31 | 2020-04-28 | 山东博科生物产业有限公司 | Direct bilirubin detection kit |
| CN112986584A (en) * | 2021-02-23 | 2021-06-18 | 潍坊泽成生物技术有限公司 | Method for making total bilirubin determination reagent kit |
| CN114277088A (en) * | 2021-12-02 | 2022-04-05 | 深圳市锦瑞生物科技股份有限公司 | Total bilirubin determination reagent, preparation method of reagent ball and determination chip |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109813918A (en) * | 2019-01-11 | 2019-05-28 | 河北省医疗器械与药品包装材料检验研究院(河北省医疗器械技术审评中心) | A kind of total bilirubin determination reagent kit |
| CN109813918B (en) * | 2019-01-11 | 2021-04-09 | 河北省药品医疗器械检验研究院 | Total bilirubin determination kit |
| CN111077322A (en) * | 2019-12-31 | 2020-04-28 | 山东博科生物产业有限公司 | Direct bilirubin detection kit |
| CN112986584A (en) * | 2021-02-23 | 2021-06-18 | 潍坊泽成生物技术有限公司 | Method for making total bilirubin determination reagent kit |
| CN114277088A (en) * | 2021-12-02 | 2022-04-05 | 深圳市锦瑞生物科技股份有限公司 | Total bilirubin determination reagent, preparation method of reagent ball and determination chip |
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