CN106404686B - A kind of serum total bilirubin of antiheparin (Vanadic acid oxidation) detection kit - Google Patents
A kind of serum total bilirubin of antiheparin (Vanadic acid oxidation) detection kit Download PDFInfo
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- CN106404686B CN106404686B CN201610735492.8A CN201610735492A CN106404686B CN 106404686 B CN106404686 B CN 106404686B CN 201610735492 A CN201610735492 A CN 201610735492A CN 106404686 B CN106404686 B CN 106404686B
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- 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 present invention relates to serum total bilirubin (Vanadic acid oxidation) detection technique fields, in particular to a kind of serum total bilirubin detection reagent, contain disodium hydrogen phosphate-citrate buffer solution, cetyl trimethylammonium bromide, fatty alcohol polyoxyethylene ether, alkyl phenol polyoxyethylene ether (OP), PEG 8000, lauryl sodium sulfate in reagent R1, contain phosphate buffer, disodium ethylene diamine tetraacetate and sodium metavanadate in reagent R2, the reagent is suitble to match with various automatic clinical chemistry analyzers, and testing result is not influenced by heparin.
Description
Technical field
The present invention relates to serum total bilirubin (Vanadic acid oxidation) detection technique fields, in particular to a kind of to be suitble to entirely certainly
Serum total bilirubin (Vanadic acid oxidation) detection reagent of Automatic Biochemical Analyzer.
Background technique
Total bilirubin isBilirubin directWith the summation of both indirect bilirubins.Total bilirubin source mainly has: 1. 80%~
85% bilirubin is disintegrated from the red blood cell of aging;2. about 15% by red blood cell quilt in marrow still immature in hematopoiesis
Destroy (ineffectivity RBC acceptor garland rate in marrow) and formed;3. heme-containing protein (hemoprotein) is come from a small quantity, such as flesh
The destruction of Lactoferrin, peroxidase, cytochromes etc. is decomposed.
Total bilirubin is mainly used to whether diagnosis has whether liver diseases or biliary tract are abnormal, 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;Greater than 340 μm ol/L are then severe jaundice.
Clinically common serum total bilirubin detection method has heavy nitrogen, Bilirubin Oxidase method and vanadate oxidation
Method.Heavy nitrogen reagent has that higher experiment sensitivity, reagent price are cheap, detection operating method is simple, but repeatability is unstable;
Bilirubin Oxidase method has many advantages, such as the good, high sensitivity of specificity, reproducible, easy to operate, but due to reagent price compared with
Height is not widely used yet at present.Later with vanadate method detection blood mesobilirubin method appearance and at home by
It gradually promotes, the evaluation in clinic is also more, and the good specificity of owner and stability have obtained the good of clinical use person
It comments, but as the gradually popularization of clinical use is found, the plasma sample obtained in being directed to clinic using heparin processing,
When detecting total bilirubin using vanadate method, cause testing result inaccurate, response curve exception and testing result is abnormal is held
It is relatively low or even negative value occur to easily lead to testing result, this in clinic popularization and use cause very big puzzlement.
Summary of the invention
It is directed to serum total bilirubin detection, the present invention provides a kind of serum total bilirubin of antiheparin (vanadate methods)
Detection kit, compared with common detection methods, testing result is not influenced the kit by heparin, and using simply, conveniently, is had
There are good accuracy, repeatability and good detection range, is conducive to the popularization and application of reagent clinically.
The present invention is achieved by the following measures:
Original cationic surfactant cetyl trimethylammonium bromide is replaced to make using novel surfactant
For accelerator, the total hydrogen bond of unconjugated bilirubin is destroyed, is then fully exposed the bilirubin after accelerating the failure, in PH=3
Near, vanadate is acted on into sample, the total bilirubin in sample is oxidized to biliverdin, and yellow specific to bilirubin subtracts
It is few, its difference that front and back absorbance is aoxidized by vanadate is detected at wavelength 450nm, variation degree contains with the TBIL's in sample
It measures directly proportional.
A kind of total bilirubin detection reagent, including reagent R1, R2, the composition of described reagent R1, R2 are as follows:
Reagent R1:
Disodium hydrogen phosphate-benzoic acid buffer (pH2.9) 0.1mol/L
Fatty alcohol polyoxyethylene ether 5g/L-10 g/L
4 g/L -8g/L of alkyl phenol polyoxyethylene ether (OP)
PEG 8000 1%-5.0%
Qula leads to -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
Disodium ethylene diamine tetraacetate 5g/L-10g/L
Tetrasodium ethylenediamine tetraacetate 1g/L-2g/L
Sodium metavanadate 4mmol/L
R1:R2=4:1, R1 dosage are 240 μ l to the reagent when in use.
The beneficial effects of the present invention are:
1, the present invention is limited has selected benzoic acid as main buffer, both vanadate can be promoted to the oxygen of bilirubin
Change effect, and corrosion-resistant can be played the role of to reaction system；
2, the present invention in selected fatty alcohol polyoxyethylene ether, alkyl phenol polyoxyethylene ether (OP), polyethylene glycol etc. non-from
Accelerator of the subtype surface-active as bilirubin, so as to avoid the poly- of cationic surfactant and macromolecular heparin substance
It closes, and can be good at playing the role of sufficiently accelerating, three kinds of surfactant tool synergistic effects；
3, in reagent while adding disodium ethylene diamine tetraacetate, it is added to a small amount of tetrasodium ethylenediamine tetraacetate chela
Mixture can play a role different heavy metal ion, and tetrasodium ethylenediamine tetraacetate is able to suppress vanadate to other
The oxidation of substance, to improve the specificity of testing result.
4, the bright Qula that is added in reagent 1 of this law leads to -305 surfactants, can be improved the clarification journey of reaction system
Degree, improves the precision of product.
5, preferred sodium fluoride is used as ionic equilibrium agent in the present invention, and adjustment reaction process intermediate ion concentration is to reaction
Influence.
Detailed description of the invention
Fig. 1 is the correlation curve figure of two kinds of reagents；
Fig. 2 is reagent stability of the present invention;
1 reagent of Fig. 3 embodiment detects operating method
2 reagent interference free performance of Fig. 4 embodiment compares；
1 reagent of Fig. 5 embodiment and market is common and the serum total bilirubin determination kit contrasting detection result that gets the nod.
Specific embodiment
Invention is further explained combined with specific embodiments below:
Embodiment 1
Disodium hydrogen phosphate-benzoic acid buffer (pH2.9) 0.1mol/L
Fatty alcohol polyoxyethylene ether 5g/L-10 g/L
4 g/L -8g/L of alkyl phenol polyoxyethylene ether (OP)
PEG 8000 1%-5.0%
Qula leads to -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
Disodium ethylene diamine tetraacetate 5g/L-10g/L
Tetrasodium ethylenediamine tetraacetate 1g/L-2g/L
Sodium metavanadate 4mmol/L
The application method of the present embodiment reagent:
Serum total bilirubin (Vanadic acid oxidation) detection reagent of the present embodiment description, when in use using full-automatic raw
Change analyzer, such as 7180 fully-automatic analyzer of Hitachi, is measured using Two point end assay.The ratio of sample and reagent is set
It is set to 9:240:60, Detection wavelength 450nm, places distilled water, standard items and sample in the corresponding position of sample disc, operates
Such as Fig. 3.
Embodiment 2
Interference test: taking fresh mix serum, be divided into 2 equal portions, and every equal portions are then separated into 5 equal portions, is added different
Interfering substance, so that its concentration in serum is reached the requirement of Fig. 4.Then 1 gained reagent of embodiment is used respectively, it is normal with market
See and the content of the serum total cholesterol reagent approved comparative determination serum total cholesterol simultaneously, control group measurement result and is added
The measurement result of each group is shown in Fig. 4 after disturbance substance.Relative deviation (%)=(measurement mean value-check sample of interference sample
Measure mean value)/check sample measurement mean value × 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 significantly interfere with 1 reagent test result of embodiment.Compared with results of comparison, reagent of the present invention
Antiheparin ability is obvious, upper consistent with reference product in the detection of other interfering substances, illustrate the reagent of embodiment 1 accuracy with
It is up to standard on anti-interference ability.
Embodiment 3
Correlation experiment: reagent preparation, the common State Food and Drug Administration with market are formulated using embodiment 1
Serum total bilirubin (Vanadic acid oxidation) kit for certain company approved carries out control test, while having detected 20 clinics
Serum sample, testing result are as shown in Figure 5.And the correlation curve (as shown in Figure 1) of two kinds of reagents is obtained, it is tied by detection
Fruit shows that the related coefficient of two kits is 0.999, illustrates that the two has great correlation.
Calibration object and quality-control product used is tested to be respectively as follows:
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 range: 23.2 ~ 35.4 μ
mol/L。
Embodiment 4
Reagent stability verification test:
Gained detection reagent in the embodiment of the present invention 1 ~ 3 is taken into a kind of commercially available serum total bilirubin (vanadic acid as test group
Salt oxidizing process) detection kit as a control group, test group asks for identical two parts with compareing every group of group reagent, and portion does 15
Its corkage stability test, reagent is placed in 2-8 DEG C of refrigerating box of instrument and (is not taken out within 15 days), opened as 15 days
Detection of Stability；Another does 37 DEG C of heat stability test tests, and closing is placed in 37 DEG C of thermostat water baths (only to be examined daily
It is taken out when survey, after detection, still sealing is put back in 37 DEG C of water-baths, and continuous 7 days), as 7 days 37 DEG C of thermal stability
Verifying.Reagent is detected, and in instrument on 7180 automatic clinical chemistry analyzer device of Hitachi according to following Fig. 3 method simultaneously
Standard curve is established on device.Freeze-dried powder quality-control product is taken, after being uniformly dissolved, is divided into 15 parts, -20 DEG C store, daily Quality Control one
It is a, and tracing detection is as a result, it opens tracking and monitoring trend such as Fig. 2.
Claims (2)
1. a kind of serum total bilirubin Vanadic acid oxidation detection kit of antiheparin, it is characterised in that by reagent R1, R2 group
At wherein R1 includes
Disodium hydrogen phosphate -2.9 0.1mol/L of benzoic acid pH of buffer,
Fatty alcohol polyoxyethylene ether 5g/L-10g/L,
Alkyl phenol polyoxyethylene ether OP 4g/L-8g/L,
PEG 8000 1%-5.0%,
Qula lead to -305 2.5g/L-5g/L,
Sodium fluoride 1.0g/L-2.0g/L,
Tetrasodium ethylenediamine tetraacetate 1g/L-2g/L；
R2 includes
Phosphate buffer pH7.0 10mmol/L,
Disodium ethylene diamine tetraacetate 5g/L-10g/L,
Tetrasodium ethylenediamine tetraacetate 1g/L-2g/L,
Sodium metavanadate 4mmol/L.
2. a kind of serum total bilirubin Vanadic acid oxidation detection kit of antiheparin according to claim 1, special
Sign is that R1:R2=4:1, R1 dosage are 240 μ l to the reagent when in use.
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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 true CN106404686B (en) | 2019-03-29 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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