CN107255622A - A kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay - Google Patents
A kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay Download PDFInfo
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- CN107255622A CN107255622A CN201710521939.6A CN201710521939A CN107255622A CN 107255622 A CN107255622 A CN 107255622A CN 201710521939 A CN201710521939 A CN 201710521939A CN 107255622 A CN107255622 A CN 107255622A
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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Abstract
The application provides a kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay, including fibrin ferment, fibrin ferment chromophoric substrate, human plasma standard items and dilution containing dabigatran；The working concentration of fibrin ferment is 0.1~2U/ml；The working concentration of fibrin ferment Chromogenic assay is 0.05~1mmol/L.Detection mechanism is that fibrin ferment adds sample blood plasma, and its chromophoric substrate of Thrombin cleavage obtains absorbance signal.And the dabigatran in blood plasma can suppress fibrin ferment, so within the scope of certain, the content and absorbance signal of dabigatran are negatively correlated.The kit is based on fibrin ferment Chromogenic assay method and realizes dabigatran changes of contents in monitoring blood plasma.The kit, which is realized, quick and precisely detects dabigatran content, and dabigatran is in the range of 0~500ng/ml, R >=0.99, and preferably, the kit relative deviation is less than 1% to linear relationship, and accuracy is high.
Description
Technical field
The invention belongs to technical field of medical examination, and in particular to one kind detects Da Bijia based on fibrin ferment Chromogenic assay
The kit of group's content.
Background technology
Dabigatran etcxilate (dabigatran etexilate) is a kind of new oral anticoagulation, by directly suppressing blood coagulation
Enzyme (clotting factor FIIa) plays anticoagulation, is formed available for prevention marrow joint/postoperative VTE of knee prosthesis,
Or prevention auricular fibrillation causes cerebral apoplexy etc..Although the medicine of anticoagulation has a lot, such as warfarin, dabigatran etcxilate
With obvious advantage：Rapid-action, anti-freezing curative effect is expectable etc., but such as all anti-coagulants, dabigatran etcxilate can cause
Blood event.At present also it is untapped go out for dabigatran etcxilate specific antagonists.And clinically take the trouble of dabigatran etcxilate
Once there is massive haemorrhage in person, or needs the invasive processing of the latter of performing the operation, and can typically take the measure of urgent reverse anticoagulating active
To tackle.This method can also bring the dual injury of physiology and psychology not only bad for bleeding is prevented in advance to patient, because
This, has had a strong impact on the health of patient and has recovered.
Although dabigatran etcxilate is compared with higher security compared with warfarin with Enoxaparin, the medicine is by hospital
With patient in use, with the increase of number of users, it has been found that monitoring and adjustment dosage to dabigatran haemoconcentration are
It is necessary, severe haemorrhage risk can be greatly lowered its close monitoring, so as to improve the security of medication.Activated partial coagulates
Blood movable enzyme time (aPTT) is the screening test for determining Inner sources property blood coagulation system, can also be used as intrinsic pathway clotting factor
Quantitative test.This method can for detect dabigatran blood content；Its principle is that test plasma is added into part blood coagulation to live
Enzyme solutions, fibrinogen is changed into insoluble fibrin in calcium ion presence, determines the time needed for solidification, as treats
Survey blood plasma activated partial thromboplastin time；Having used the blood plasma activated partial thromboplastin time of dabigatran can extend, this
Method can be used to monitor the blood content of dabigatran.Activated partial thromboplastin time (aPTT) is to determine Inner sources property blood coagulation system
Screening test, can also as intrinsic pathway clotting factor quantitative test；Clotting time, i.e., lived from addition part blood coagulation
Enzyme solutions are the basis entirely tested to the time of the clotting of plasma, therefore the clotting time must be accurate, and otherwise experimental result is difficult to
Accurately, partial thromboplastin solution is added in test plasma, have activated whole piece intrinsic coagulation pathway, cause the clotting of plasma.It is whole
The clotting factor that bar intrinsic coagulation pathway is involved is numerous, and the disturbing factor being subject in this way is more, the blood of dabigatran
Liquid concentration and the coefficient correlation of activated partial thromboplastin time be not strong, it is difficult to is accurately quantified.
The content of the invention
In view of this, it is an object of the invention to provide rapid accurate, it is possible to achieve the dabigatran of automatic measurement contains
The kit of amount.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay, including blood coagulation
Enzyme, fibrin ferment chromophoric substrate, human plasma standard items and dilution containing dabigatran；
The working concentration of fibrin ferment is 0.1~2U/ml；
The working concentration of fibrin ferment chromophoric substrate is 0.05~1mmol/L.
It is preferred that, the working concentration of the fibrin ferment is 0.2~1U/ml.
It is preferred that, the working concentration of the fibrin ferment chromophoric substrate is 0.1~0.5mmol/L.
It is preferred that, the fibrin ferment chromophoric substrate is H-D-CHG-Ala-Arg-pNA2AcOH, Tos-Gly-Pro-
Arg-pNA·AcOH、H-D-CHG-But-Arg-pNA·2AcOH、H-D-CHG-Pro-Arg-pNA·2AcOH、H-D-CHA-
Ala-Arg-pNA·2AcOH、H-D-CHA-Gly-Arg-pNA·2AcOH、CH3OCO-Gly-Pro-Arg-pNA·AcOH、H-
β-Ala-Gly-Arg-pNA2AcOH, H-D-Phe-Pip-Arg-pNA2HCl, pyroGlu-Pro-Arg-pNAHCl or
H-D-Ala-Pro-Arg-pNA·2HCl。
It is preferred that, the component of the dilution includes buffer solution, inorganic salts, stabilizer, surfactant and preservative.
It is preferred that, the buffer solution is TRIS buffer or phosphate buffer；The pH of the buffer solution
It is worth for 7.2~7.8.
It is preferred that, the inorganic salts are sodium chloride or potassium chloride；The mass concentration of the inorganic salts is preferably 0.15~
0.25mol/L；
It is preferred that, the surfactant is PEG-8 000 or Tween-20；The quality of the surfactant is dense
Spend for 0.05%~0.15%.
It is preferred that, the stabilizer is calf serum or casein；The mass concentration of the stabilizer be 0.08%~
0.15%；
It is preferred that, the preservative is Proclin 300 or Sodium azide；The mass concentration of the preservative is preferably 0.1
~1mg/ml.
This application provides a kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay, including blood coagulation
Enzyme, fibrin ferment chromophoric substrate, human plasma standard items and dilution containing dabigatran；The working concentration of fibrin ferment be 0.1~
2U/ml；The working concentration of fibrin ferment chromophoric substrate is 0.05~1mmol/L.The detection mechanism of the kit is that fibrin ferment adds
Chromophoric substrate can be acted on by entering after sample blood plasma, signal is produced by cracking its chromophoric substrate.And the dabigatran meeting in blood plasma
Suppress fibrin ferment, dabigatran content is higher, and the suppression to fibrin ferment is stronger, and obtained absorbance signal is weaker.So one
Within the scope of fixed, the content of dabigatran and the signal of absorbance are negative correlativing relations, and it is fixed to be reached according to standard curve
Measure the purpose of detection.Kit of the present invention is based on fibrin ferment Chromogenic assay and realizes that dabigatran contains quantitative change in monitoring blood plasma
Change, the kit, which is realized, quick and precisely detects dabigatran content, and research finds that dabigatran concentration is in 0~500ng/ml
When, R >=0.99, preferably, while the kit relative deviation is less than 1%, accuracy is preferable for linear relationship.
Meanwhile, when the kit that the present invention is provided is by multiple measurement, CV values are less than 2%, and repeatability is preferably.The reagent
Box can detect as little as 15ng/ml dabigatran.
Brief description of the drawings
Fig. 1 is the canonical plotting drawn in embodiment 3.
Embodiment
The invention provides a kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay, including blood coagulation
Enzyme, fibrin ferment chromophoric substrate, human plasma standard items and dilution containing dabigatran；
The working concentration of fibrin ferment is 0.1~2U/ml；
The working concentration of fibrin ferment chromophoric substrate is 0.05~1mmol/L.
The invention provides a kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay, research hair
Existing, dabigatran concentration is in 0~500ng/ml, R >=0.99, and linear relationship is preferable, while the kit relative deviation is small
In 1%, accuracy is preferable.
A kind of kit based on fibrin ferment Chromogenic assay detection dabigatran content that the present invention is provided includes blood coagulation
Enzyme.The working concentration of the fibrin ferment is 0.1~2U/ml, more preferably preferably 0.2~1U/ml, 0.5U/ml.The blood coagulation
The source of enzyme is not particularly limited, using fibrin ferment well-known to those skilled in the art.In the present invention, the fibrin ferment
Purchased from sigma (Sigma).
A kind of kit based on fibrin ferment Chromogenic assay detection dabigatran content that the present invention is provided includes blood coagulation
Enzyme chromophoric substrate.The working concentration of the fibrin ferment chromophoric substrate be 0.05~1mmol/L, preferably 0.1~0.5mmol/L,
More preferably 0.3mmol/L.The fibrin ferment chromophoric substrate is H-D-CHG-Ala-Arg-pNA2AcOH, Tos-Gly-Pro-
Arg-pNA·AcOH、H-D-CHG-But-Arg-pNA·2AcOH、H-D-CHG-Pro-Arg-pNA·2AcOH、H-D-CHA-
Ala-Arg-pNA·2AcOH、H-D-CHA-Gly-Arg-pNA·2AcOH、CH3OCO-Gly-Pro-Arg-pNA·AcOH、H-
β-Ala-Gly-Arg-pNA2AcOH, H-D-Phe-Pip-Arg-pNA2HCl, pyroGlu-Pro-Arg-pNAHCl and
One or more in H-D-Ala-Pro-Arg-pNA2HCl.The source of the fibrin ferment chromophoric substrate is not particularly limited,
Using fibrin ferment chromophoric substrate well-known to those skilled in the art.
A kind of kit based on fibrin ferment Chromogenic assay detection dabigatran content that the present invention is provided includes dilution
Liquid.The effect of the dilution is that fibrin ferment and fibrin ferment chromophoric substrate are diluted, and obtains fibrin ferment and fibrin ferment color development
Substrate solution.
In the present invention, the component of the dilution includes buffer solution, inorganic salts, stabilizer, surfactant and preservative.
The buffer solution is preferably TRIS buffer or phosphate buffer, and more preferably trishydroxymethylaminomethane delays
Fliud flushing；The pH value of the buffer solution is 7.2~7.8, more preferably 7.5.The inorganic salts are preferably sodium chloride or potassium chloride, more
Preferably sodium chloride；The mass concentration of the inorganic salts is preferably 0.15~0.25mol/L, more preferably 0.2mol/L；It is described
Surfactant is preferably PEG-8 000 or Tween-20, more preferably PEG-8 000；The surfactant
Mass concentration is preferably 0.05%~0.15%, and more preferably 0.1%.The stabilizer is preferably calf serum or casein,
More preferably calf serum；The mass concentration of the stabilizer is preferably 0.08%~0.15%, and more preferably 0.1%；It is described
Preservative is preferably Proclin 300 or Sodium azide；The mass concentration of the preservative is preferably 0.1~1mg/ml, more preferably
For 0.5mg/ml.
It is a kind of to detect the kit of dabigatran content in detection Da Bijia based on fibrin ferment Chromogenic assay in the present invention
Application in group's content, comprises the following steps：
1) fibrin ferment and fibrin ferment chromophoric substrate are diluted with dilution, obtains the molten of fibrin ferment and fibrin ferment chromophoric substrate
Liquid；
2) testing sample is mixed with thrombin solution, be incubated, the mixed liquor after being incubated；
3) after the mixed liquor after incubation being mixed with the solution of fibrin ferment chromophoric substrate, is incubated, read in testing sample and send out
The signal intensity of color substrate；
4) obtained calculating signal intensity and the standard curve of concentration known standard items are compared, obtains Da Bijia in blood
Group's content.
In the present invention, the step 2) in the volume ratio of testing sample and thrombin solution be 1~2:1, more preferably 1:
1。
The step 2) in be incubated temperature be preferably 37 DEG C.The time of the incubation is preferably 2 minutes.
In the present invention, the volume ratio of mixed liquor and the solution of fibrin ferment chromophoric substrate is 1~2 after the incubation:1, it is more excellent
Elect 1 as:1.
In the present invention, the step 3) in the Detection wavelength of signal intensity be 405nm.
The present invention can also detect other direct thrombin inhibitors except that can detect dabigatran content.
In the present invention, method of the method for detecting other thrombin inhibitors with detection dabigatran.
Fibrin ferment Chromogenic assay is based on to one kind that the present invention is provided with reference to embodiment and detects dabigatran content
Kit be described in detail, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
Concentration is 20mmol/L Tris buffer solutions, adds salt acid for adjusting pH value to 7.5；Add sodium chloride, poly- second
It is respectively sodium chloride 0.2mol/L that final concentration is obtained after the calf serum of glycol -8000 and Sodium azide, stirring, and polyethylene glycol -
80000.1%, calf serum 0.1%, Sodium azide 0.5mg/ml dilution.
Take fibrin ferment to be dissolved in dilution, form working concentration for 0.1U/ml, 0.2U/ml, 0.5U/ml, 1U/ml and 2U/
Ml thrombin solution.
Take the chromophoric substrate H-D-Phe-Pip-Arg-pNA2HCl of fibrin ferment to be dissolved in dilution, form working concentration
For the chromophoric substrate solution of 0.05mmol/L, 0.1mmol/L, 0.3mmol/L, 0.5mmol/L and 1mmol/L fibrin ferment.
Embodiment 2
Sample detection：
100 μ L samples are taken to be well mixed with 100 μ l thrombin solutions, 37 DEG C are incubated 2 minutes, then add 200 μ l blood coagulations
The chromophoric substrate solution of enzyme, 37 DEG C of reactions were in 405nm wavelength measurements absorbance 5 minutes.The color development of thrombin solution and fibrin ferment
The working concentration selection of substrate solution is as shown in table 2.Log, calculates signal intensity, and standard curve is compared and obtained
Dabigatran content.
The result that the detection dabigatran of table 1 is determined
As can be seen from Table 1, such scheme can determine the changes of contents of dabigatran exactly, and wherein fibrin ferment works
The scheme Detection results that concentration is 0.5U/ml and the working concentration of the chromophoric substrate of fibrin ferment is 0.3mmol/L are optimal.
Embodiment 3
The preparation of dabigatran standard items：Prepare the human plasma standard of the known dabigatran concentration of multiple various concentrations
Product, its concentration is respectively 0ng/ml, 15ng/ml, 30ng/ml, 60ng/ml, 120ng/ml, 250ng/ml, 500ng/ml.
Standard curve making：
(1) take 100 μ L titers to be well mixed with 100 μ L thrombin solutions, and be incubated 2 minutes, the temperature for being incubated and reacting
Degree is 37 DEG C.
(2) then add 200 μ L fibrin ferments chromophoric substrate solution, be well mixed, in 405nm wavelength measurements absorbance 5
Minute.
(3) according to dabigatran titer gradient concentration and corresponding light absorption value, standard curve is drawn using linear equation,
See Fig. 1, its calibration curve formula is y=-0.7441x+0.2067 (R2=0.9927).
The detection dabigatran of table 2 determines the kit range of linearity
| Dabigatran concentration (ng/ml) | 0 | 15 | 30 | 60 | 120 | 250 | 500 |
| Dabigatran concentration (ng/l) | 0 | 0.015 | 0.03 | 0.06 | 0.12 | 0.25 | 0.5 |
| 405nm absorbances | 1.679 | 1.588 | 1.507 | 1.432 | 1.291 | 1.014 | 0.699 |
As shown in Table 2, dabigatran concentration is in 0~500ng/ml range contents, with preferable linear relationship, in this model
The content of dabigatran in blood plasma can be accurately detected in enclosing.
Embodiment 4
According to the detection method in embodiment 3, fibrin ferment working concentration is the work of the chromophoric substrate of 0.3U/ml and fibrin ferment
Make the scheme that concentration is 0.5mmol/L to detect different gradient dabigatran concentration, obtained after obtaining absorbance, calculating
The detection sensitivity of this kit.According to national standard, sensitivity is the slope of standard curve, i.e., with concentration known or activity
Sample test kit, records the absorbance that is produced in the case where kit provides parameter and changes, the slope of its standard curve for-
0.7441log (absorbance)/(ng/l).
The detection dabigatran of table 3 determines kit sensitivity
| Dabigatran concentration (ng/ml) | 0 | 15 | 30 | 60 | 120 | 250 | 500 |
| 405nm absorbances | 1.679 | 1.588 | 1.507 | 1.432 | 1.291 | 1.014 | 0.699 |
As shown in Table 3, the kit that the present invention is provided can detect as little as 15ng/ml dabigatran.
Embodiment 5
The concentration of dabigatran standard items is examined for many times for 30ng/ml and 250ng/ml according to the detection method of embodiment 3
Absorbance is surveyed, 4 are the results are shown in Table.
The detection dabigatran of table 4 determines kit repeatability
As shown in Table 4, when the kit that the present invention is provided passes through repeated detection, CV values are less than 2%, and repeatability is very good.
Embodiment 6
According to the detection method in embodiment 3, snake vein enzyme working concentration is the chromophoric substrate of 0.2U/ml and fibrin ferment
Working concentration detects for 1mmol/L scheme to different diluent, is utilized the standard curve of different diluent.
Table 5 detects influence of the different diluent to kit
As can be seen from Table 5, the influence that has different degree of the different diluent species to the standard curve of kit, its
The standard curve R of middle carbonate buffer solution2It is worth for 0.9727, the R of the standard curve of borate buffer solution2It is worth for 0.9759, and three
The standard curve R of hydroxymethyl aminomethane buffer solution and phosphate buffer2Value is all higher than 0.99, therefore, trihydroxy methyl amino
Aminomethane buffer or phosphate buffer are conducive to improving the detection accuracy and accuracy of kit.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay, including fibrin ferment, fibrin ferment color development
Substrate, human plasma standard items and dilution containing dabigatran；
The working concentration of fibrin ferment is 0.1~2U/ml；
The working concentration of fibrin ferment chromophoric substrate is 0.05~1mmol/L.
2. kit according to claim 1, it is characterised in that the working concentration of the fibrin ferment is 0.2~1U/ml.
3. kit according to claim 1, it is characterised in that the working concentration of the fibrin ferment chromophoric substrate is 0.1
~0.5mmol/L.
4. kit according to claim 1, it is characterised in that the fibrin ferment chromophoric substrate is H-D-CHG-Ala-
Arg-pNA·2AcOH、Tos-Gly-Pro-Arg-pNA·AcOH、H-D-CHG-But-Arg-pNA·2AcOH、H-D-CHG-
Pro-Arg-pNA·2AcOH、H-D-CHA-Ala-Arg-pNA·2AcOH、H-D-CHA-Gly-Arg-pNA·2AcOH、
CH3OCO-Gly-Pro-Arg-pNA·AcOH、H-β-Ala-Gly-Arg-pNA·2AcOH、H-D-Phe-Pip-Arg-pNA·
2HCl, pyroGlu-Pro-Arg-pNAHCl or H-D-Ala-Pro-Arg-pNA2HCl.
5. kit according to claim 1, it is characterised in that the component of the dilution include buffer solution, inorganic salts,
Stabilizer, surfactant and preservative.
6. kit according to claim 5, it is characterised in that the buffer solution is TRIS buffer
Or phosphate buffer；The pH value of the buffer solution is 7.2~7.8.
7. kit according to claim 5, it is characterised in that the inorganic salts are sodium chloride or potassium chloride；The nothing
The molar concentration of machine salt is 0.15~0.25mol/L.
8. kit according to claim 5, it is characterised in that the surfactant is PEG-8 000 or told
Temperature -20；The mass percent of the surfactant is 0.05%~0.15%.
9. kit according to claim 5, it is characterised in that the stabilizer is calf serum or casein；It is described
The mass percent of stabilizer is 0.08%~0.15%.
10. kit according to claim 5, it is characterised in that the preservative is Proclin300 or Sodium azide；Institute
The mass concentration for stating preservative is 0.1~1mg/ml.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710521939.6A CN107255622A (en) | 2017-06-30 | 2017-06-30 | A kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay |
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|---|---|---|---|
| CN201710521939.6A CN107255622A (en) | 2017-06-30 | 2017-06-30 | A kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay |
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| CN114277089A (en) * | 2021-12-24 | 2022-04-05 | 北京赛科希德科技股份有限公司 | Dabigatran detection reagent and kit |
| CN114958963A (en) * | 2022-07-29 | 2022-08-30 | 深圳传世生物医疗有限公司 | Anticoagulant drug detection kit acting on thrombin and application thereof |
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| CN114958963B (en) * | 2022-07-29 | 2023-09-05 | 深圳传世生物医疗有限公司 | Anticoagulation medicine detection kit acting on thrombin and application thereof |
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