CN106153612A - Antithrombin activity detectable and its preparation method and application - Google Patents
Antithrombin activity detectable and its preparation method and application Download PDFInfo
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- CN106153612A CN106153612A CN201610435113.3A CN201610435113A CN106153612A CN 106153612 A CN106153612 A CN 106153612A CN 201610435113 A CN201610435113 A CN 201610435113A CN 106153612 A CN106153612 A CN 106153612A
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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
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
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- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
Abstract
The invention discloses antithrombin activity detectable, including reagent 1 and reagent 2, wherein: reagent 1 is the chromophoric substrate of FXa, reagent 2 is the diluent containing FXa and heparin；Wherein the chromophoric substrate of FXa is selected from following: CH3SO2‑D‑Leu‑Gly‑Arg‑p‑nitroanilide·AcOH；CH3OCO‑D‑CHG‑Gly‑Arg‑p‑nitroanilide·AcOH；CH3OCO‑D‑CHA‑Gly‑Arg‑p‑nitroanilide·AcOH；Benzoyl‑Ile‑Glu‑Gly‑Arg‑p‑nitroanilide·HCl；Suc‑Ile‑Glu(γ‑Piperidyl)‑Gly‑Arg‑p‑nitroanilide·HCl；N‑α‑Z‑D‑Arg‑Gly‑Arg‑p‑nitroanilide·2HCl；Boc‑D‑Arg‑Gly‑Arg‑p‑nitroanilide·2HCl；Acetyl‑D‑Arg‑Gly‑Arg‑p‑nitroanilide·2HCl；4‑Nz‑D‑Arg‑Gly‑Arg‑p‑nitroanilide·2HCl；4‑Mbs‑D‑Arg‑Gly‑Arg‑p‑nitroanilide·2HCl.The present invention detects stability and reproducible, has the highest sensitivity and accuracy, can the activity of accurate response antithrombase, and find the abnormal patient of internal antithrombin activity, thus provide foundation for treatment.
Description
Technical field
The invention belongs to Biological Detection technical field, relate to a kind of antithrombin activity detectable and preparation method thereof
And application.
Background technology
One, hemostasis (blood coagulation) mechanism of human body
Hemostasis (blood coagulation) mechanism is a very important self-protection function of human body, and hemostasis (blood coagulation) mechanism includes coagulating
Blood and two aspects of anticoagulant, dynamic equilibrium between the two is that normal body maintains internal blood flow regime and prevents blood loss
Key, it can carry out the wound of sealing blood vessels when vascular accident breakage by hematoblastic cohesion thus prevent substantial amounts of blood
Run off.Hemostasis (blood coagulation) process is the process that a series of thrombin is deactivated by sequential enzymatic, and hemostasis (blood coagulation) process is the most lifelong
Become thrombin, form fibrin clot.Especially hemostasis (blood coagulation) process is a process being refined regulation and control, hematoblastic solidifying
The poly-wound that a certain amount should be had to ensure to close blood vessel can not condense again and too much block blood vessel on the contrary.The normal of body stops
Blood coagulation, depends on complete wall structures and function, effective platelet quality and quantity, and normal blood plasma coagulates
Blood factor activity.
The process of the control accurate of such a complexity is divided into: initial stage hemostasis, second phase hemostasis, the hemostasis of three phases, be little by blood
Plate, blood vessel wall and a series of thrombin and fibrinolytic factor jointly participate in and have interacted.Initial stage stops blooding
Relate to the adhesion on damaged blood vessels surface of the contraction of damaged blood vessels, the exposure of subendothelial collagen tissue and platelet, gathering and
Formation initial stage tampon.Bring owing to they are also insufficiently resistant to the blood produced shearing force that flows in the blood vessel in intensity
Impact, old friend's body hemostasis (blood coagulation) mechanism also need to fibrin condensation product further combined with effect and Stabilization ability
Enough complete to close the task of the wound of blood vessel.Second phase hemostasis refers to that the position forming initial stage tampon forms fiber further
The process of fibrin clot.The generation of fibrin condensation product is a complicated process.Vessel wall injury breakage causes a series of
The activation of thrombin, has activated the factor (i.e. thrombin) X by the transmission of blood coagulation waterfall, and activation factor X is (hereinafter referred to as
FXa) it is thrombin prothrombin activation further.Thrombin is a kind of multi-functional protease, and it is non-in hemostasis
The normally off key, is a thrombin being in core status.It is converted into fibrin Fibrinogen, and fibrin is subsequently
Polymerization forms fibrin condensation product.Fibrin condensation product passes through and albumen on platelet aggregates surface of cell membrane
Effect is stablized them and is increased their intensity further.The most just can close the wound more loss of anti-Hemostatic Oral Liquid of blood vessel.
When the wound healing of blood vessel is repaired, the plasmin solution preocess startup in blood removes these fibrin condensation products and keeps away
Exempt from blood vessel blockage and other harm.The hemostasis of three phases is by platelet aggregation compound, fiber protein yarn and the erythrocyte institute being absorbed in
The loose net of composition, defines firm sludged blood by this process.Under normal circumstances, when blood vessel does not has injured,
Blood system there are some mechanism cause fibrin condensation product to form even blood to prevent the improper startup of hemostasis
Bolt.Antithrombase (also known as Antithrombin III, english abbreviation is ATIII) is exactly the most important one.
Two, the clinical meaning that in blood, antithrombase measures
Serine protease such as activation factor X in antithrombase energy enzyme anticoagulant and some other blood coagulation waterfall
(FXa), activation factor IX (FIXa) and activation factor XI (FXIa), the avtive spot of these protease and the reaction of antithrombase
Center can form complex thus the activity of these protease is suppressed.But antithrombase suppresses the efficiency of these protease
The highest, and when some glycosaminoglycans in human body, such as heparitin sulfate is when blood vessel wall and antithrombase combine, then
The conformation change that can cause antithrombase causes antithrombase to suppress the efficiency of these protease to increase about thousand times.Medically should
Heparin extract from the intestinal of cattle or pig, it is widely used as anticoagulant.It and antithrombase combine, and have and sulfuric acid liver
The effect that element is similar, also can increase antithrombase greatly and suppress the efficiency of these protease.
Antithrombase plays an important role preventing hemostatic mechanism from excessively using in thrombosis, and antithrombase disappearance meeting
Cause phlebothrombosis or pulmonary infarction disease, and antithrombase content or the low individuality of activity suffer from easy bolt disease and thrombosed
Danger is the highest.Antithrombase disappearance have genetic also have acquired: hereditary antithrombin disappearance disease 1965
Year finds in the family of Norway of a frequent generation thromboembolism.The activity of the antithrombase of normal person is defined as 100%,
Generally 75%-125% is normal range, and the antithrombin activity of this kind of patient only has the 40-70% of normal person.Acquired anti-
The reason of thrombin disappearance disease has multiple, and the antithrombase synthesis caused including hepatopathy reduces, the antithrombase stream that nephropathy causes
Lose, use the drug-induced antithrombase losses such as heparin, and the antithrombase consumption that disseminated inravascular coagulation causes.Anti-
The treatment of thrombin disappearance disease includes using heparin, China to cut down order or purification antithrombase.The most genetic or obtain
Property antithrombase disappearance disease all bring the risk of thrombosis to patient, bring the prestige being difficult to predict to their health and lives
The side of body.To the accurate detection of antithrombase then can to antithrombase disappearance disease, easy bolt disease, phlebothrombosis and pulmonary infarction disease and
Time diagnosis and treatment be significant.
Three, the influence factor that antithrombase measures
It is reported, the existing disclosed detection to antithrombase substantially has three class methods, is freezing method, immunization respectively
With the Chromogenic assay based on thrombin.Freezing method mainly compares test plasma and reference blood plasma at blood coagulation waterfall by phase
After answering reagent to activate, setting time is to measure the activity of antithrombase.Although freezing method can directly use the test plasma can also
Test plasma is heated defibrination then use, but its complex operation and also setting time variation is relatively big, be unfavorable for accurately
Measuring and calculating.Immunity class method is divided into two kinds, and the first is euzymelinked immunosorbent assay (ELISA).This method is to be coated in the hole of microtitration plate
The antibody of antithrombase, the sample blood plasma and the standard plasma that are subsequently adding different proportion dilution are hatched, and add Radix Cochleariae officinalis after cleaning
The antibody of the anti-antithrombase of peroxidase labelling, adds the substrate of horseradish peroxidase, has reacted after again cleaning
Read absorbance after Quan, obtain standard curve finally by the absorbance of standard plasma and extrapolate the antithrombase of standard plasma again
Content.Another kind of method in immunity class method is referred to as Lao Ruier rocket electrophoresis, is by the antibody of anti-antithrombase and thawing
Agar mixing then cool down plastic, carry out electrophoresis after adding sample blood plasma and standard plasma, after electrophoresis completes, antigen-antibody is multiple
Compound Coomassie blue stain detects.The antigen antibody complex that the blood plasma of different antithrombase content is formed has different
Highly, and its content and concentration are directly proportional.Containing of antithrombase in standard curve and sample blood plasma can be drawn accordingly
Amount.；Owing to above-mentioned immunity class method is manual operations, therefore differing greatly between different detection batch, complex steps, accuracy is not
Height, and automation mechanized operation can not be carried out.
3rd class method is the Chromogenic assay based on thrombin.This method be in test plasma add heparin and
The thrombin of excess is hatched, and the thrombin in reagent is formed solidifying with the antithrombase in test plasma in the presence of heparin
Hemase and the complex of antithrombase, make the activity of thrombin be suppressed.Hereafter the chromophoric substrate adding thrombin is carried out
Hatching, chromophoric substrate here is by three to four aminoacid and the 4-nitroaniline (4-covalentlying bind in end
Nitroaniline, pNA) composition, it can be by the most repressed remaining Thrombin cleavage and discharge 4-nitroaniline, this
Plant reaction and can cause the change at 405 nanometers absorbances, and the amount of the 4-nitroaniline produced is and antithrombase in sample
Activity be inversely proportional to.Therefore, the change of absorbance at measurement 405nm is utilized just can to make standard curve and extrapolate sample
The activity of middle antithrombase.
Owing to the method is highly sensitive, accuracy is good, the detection time is short, it is possible to be applicable to multiple automated analysis instrument,
And under the development of production domesticization detectable, significantly reduce Clinical detection expense, apply the most extensive in clinical practice.
Although it practice, this method is widely used in clinical practice, but mostly having testing result by blood during it measures
In slurry, the situation of multiple interfering material impact occurs.Such as, the Chromogenic assay based on thrombin is used to measure anticoagulation
Enzymatic activity, if containing thrombin inhibitor (such as hirudin, argatroban etc.) in sample, it will make testing result higher；Again
Such as the heparin co factor II of naturally occurring and Heparin-binding in blood, can anticoagulant enzymatic activity, make this method measure anticoagulation
Enzyme is affected by heparin co factor II thus is over-evaluated the activity of antithrombase in sample.Therefore, the range of this method is subject to
Certain restriction.
Four, the limitation of prior art
In order to reduce the impact of heparin co factor II, in United States Patent (USP) US5985582 and Chinese patent CN102690862A,
Disclose a kind of Antithrombin III and measure test kit (ATIII Chromogenic assay), be the Chromogenic assay based on thrombin
Being measured, test kit comprises heparin derivatives, thrombin and chromophoric substrate reagent；Described reagent is before adding sample heparin
Carried out enzymolysis, crack out one or more unsaturated disaccharidase and obtain heparin derivatives.This heparin derivatives still has
Have similar with heparin and antithrombase to combine the ability of enzyme anticoagulant, but and heparin co factor II combine and suppress solidifying
The ability of hemase is but substantially reduced.Add heparin derivatives the most in the sample rather than heparin just can be by heparin co factor II
Impact on the activity of detection antithrombase drops to the lowest.But owing to which employs the thrombin of heparin derivatives and import, and
Heparin derivatives needs the cracking from heparin to obtain, and this adds increased the cost that reagent makes, and also makes the step that reagent makes simultaneously
Cataclysm obtains comparatively laborious.Owing to this technical costs is high, therefore decreasing the consumption of reagent, reducing of volume may cause finally
During colorimetric, the numerical value of part can not show.And the prior art carries out ATIII activity on full automatic blood-coagulation instrument
Needing during detection to change a lot of parameter, inconvenient operation, the most easily there is error in result.
It addition, Chinese invention patent application CN104714036A also discloses that a kind of Antithrombin III activit assay kits
Box, it is also to have employed the Chromogenic assay based on thrombin to be measured, but the chromophoric substrate based on thrombin
There is the biggest defect in method.This method analyzes the activity of antithrombase in test plasma by adding extrinsic coagulation enzyme, due to
Another thrombin inhibitor Antithrombin Ⅲ II (HCII) outside naturally occurring antithrombase in test plasma, because of
This this method is easily caused the mensuration of antithrombase inaccurate by the interference of HCII in test plasma.Because in the blood plasma of general population
Naturally occurring Antithrombin Ⅲ II, the antithrombin activity measured in this way is inaccurate.More it is worth mentioning
It is that this method cannot be used for using hirudin (Hirudin), argatroban (Argatroban) or dabigatran
(Dabigatran) people, because these medicines are the direct inhibitor of thrombin, can make antithrombin activity detection knot
The most higher.
Summary of the invention
The problems referred to above existed for prior art, an object of the present invention is to provide a kind of antithrombin activity detection
Reagent, can avoid the interference of the anticoagulant factor such as heparin co factor II, hirudin, argatroban, dabigatran in sample to be tested,
Highly sensitive, stability and reproducible.
For achieving the above object, the technical scheme that the present invention provides is as follows:
Antithrombin activity detectable, including reagent 1 and reagent 2, it is characterised in that reagent 1 is at the bottom of the color development of FXa
Thing, reagent 2 is the diluent containing FXa and heparin.
Antithrombin activity detectable, including reagent 1, reagent 4 and reagent 3, wherein reagent 1 is the chromophoric substrate of FXa,
Reagent 4 is FXa, and reagent 3 is the diluent containing heparin.
Preferably, any in following substrate of the chromophoric substrate of above-mentioned FXa:
CH3SO2-D-Leu-Gly-Arg-p-nitroanilide·AcOH；
CH3OCO-D-CHG-Gly-Arg-p-nitroanilide·AcOH；
CH 3OCO-D-CHA-Gly-Arg-p-nitroanilide·AcOH；
Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide·HCl；
Suc-Ile-Glu(γ-Piperidyl)-Gly-Arg-p-nitroanilide·HCl；
N-α-Z-D-Arg-Gly-Arg-p-nitroanilide·2HCl；
Boc-D-Arg-Gly-Arg-p-nitroanilide·2HCl；
Acetyl-D-Arg-Gly-Arg-p-nitroanilide·2HCl；
4-Nz-D-Arg-Gly-Arg-p-nitroanilide·2HCl；
4-Mbs-D-Arg-Gly-Arg-p-nitroanilide·2HCl。
As preferably, above-mentioned antithrombin activity detectable also includes the human plasma calibration object containing antithrombase.Should
The preparation of calibration object is will to mix after normal healthy people plasma collection, more freeze-dried makes.Then WHO antithrombase is used
International standard substance blood plasma carries out traceability definite value to this calibration object.In addition, it is possible to omit these standard substance and standard curve is provided.
After being as noted previously, as heparin co factor II and Heparin-binding anticoagulant enzyme but do not suppress FXa, and the most normal
Antithrombase in using hirudin, argatroban, dabigatran or other direct thrombin inhibitor to suppress human body, by shadow
Ring testing result.
The present invention is the Chromogenic assay based on FXa, utilizes occurred conformation change after antithrombase and Heparin-binding,
Directly and effectively suppress the feature of the activity of FXa again after FXa combination, it is provided that a kind of antithrombin activity detectable, with
Chromogenic assay is made to be more widely used in antithrombin activity context of detection.Its Cleaning Principle is: in test plasma
The FXa adding heparin and excess is hatched, and the chromophoric substrate adding FXa is hatched.Wherein, heparin and test plasma
In antithrombase combine, after causing the conformation change of antithrombase, antithrombase directly and FXa combination, thus can suppress FXa
Activity.Remaining FXa cracks its chromophoric substrate and discharges 4-nitroaniline, and the amount of the 4-nitroaniline produced is and sample
In product, the activity of antithrombase is inversely proportional to.Therefore, the change of absorbance at measurement 405nm is utilized just can to make standard curve
And extrapolate the activity of antithrombase in sample.
Preferably, described heparin includes common unfraction heparin, low molecular weight heparin and fondaparin.
Preferably, mentioned reagent 1, reagent 2 or reagent 4 are corresponding solution or lyophilized preparation, preferably freeze dried powder.
Preferably, mentioned reagent 3 is corresponding solution or lyophilized preparation, preferably solution.
Preferably, the working concentration of above-mentioned chromophoric substrate is 0.1～4mM, and preferably working concentration is 0.5～3mM, optimal work
It is 2mM as concentration.
Preferably, the working concentration of above-mentioned FXa is 0.05～3 μM, and preferably working concentration is 0.1～1.5 μM, best effort
Concentration is 0.5 μM.
Preferably, the working concentration of above-mentioned heparin is 0.1～5IU/ml, and preferably working concentration is 0.5～3IU/ml, most preferably
Working concentration is 2IU/ml.
The definition of working concentration: during by any of the above-described reagent detection sample activity, the lyophilized preparation of this reagent is adopted
After redissolving with its redissolution reagent, mixing with diluted sample, in the analysis of mixtures formed, the concentration of each component is defined as it
Working concentration.
Preferably, reagent 1 is by the mix reagent comprising chromophoric substrate, buffer agent, inorganic salt, surfactant and preservative
Make.
Preferably, reagent 2 is by the mix reagent comprising FXa, heparin, buffer agent, inorganic salt, surfactant and preservative
Make.
Preferably, reagent 4 is made up of the mix reagent comprising FXa, buffer agent, inorganic salt, surfactant and preservative.
Preferably, reagent 3 is made up of the mix reagent comprising heparin, buffer agent, inorganic salt and preservative.
It is highly preferred that buffer agent delays selected from TRIS buffer (hereinafter referred to as Tris buffer) or boric acid
Rush liquid, most preferably TRIS buffer (Tris buffer).
It is highly preferred that inorganic salt is selected from sodium chloride or potassium chloride, most preferably sodium chloride.
It is highly preferred that surfactant is selected from PEG-8 000 or tween 20, most preferably PEG-8 000.
It is highly preferred that preservative may select preservative well known by persons skilled in the art, such as Proclin 300 or nitrine
Sodium, most preferably sodium azide.
Preferably, FXa is selected from any one in people, cattle, pig FXa, preferably cattle FXa.
It is a further object of the present invention to provide the preparation method of above-mentioned antithrombin activity detectable, including making respectively
Standby reagent 1, reagent 2, reagent 4 or reagent 3；Wherein:
The step preparing reagent 1 is: be dissolved in buffer agent by chromophoric substrate；Adding hydrochloric acid, regulation pH value is to 7～8；Again
It is sequentially added into inorganic salt and surfactant, stirs 10 minutes under room temperature.
Wherein, in reagent 1, final concentration or the percentage composition of each component are respectively as follows: chromophoric substrate 0.5～3mM, buffer agent
10～50mM, inorganic salt 0.1～0.3M, surfactant 0.5～1%.
The step preparing reagent 2 is: successively FXa and heparin are dissolved in buffer agent；Adding hydrochloric acid, regulation pH value is to 7～8；
Sequentially add inorganic salt and surfactant, stir 10 minutes under room temperature.
Wherein, in reagent 2, the final concentration of each component or percentage composition are respectively as follows: FXa 0.1～1.5 μMs, heparin 0.5～
3IU/ml, buffer agent 10～50mM, inorganic salt 0.1～0.3M, surfactant 0.5～1%.
The step preparing reagent 4 is: FXa is dissolved in buffer agent；Adding hydrochloric acid, regulation pH value is to 7～8；Sequentially add
Inorganic salt and surfactant, stir 10 minutes under room temperature.
Wherein, in reagent 4, the final concentration of each component or percentage composition are respectively as follows: FXa 0.1～1.5 μMs, buffer agent 10～
50mM, inorganic salt 0.1～0.3M, surfactant 0.5～1%.
The step preparing reagent 3 is: be dissolved in buffer agent by heparin；Regulation pH value is to 7～8；Add inorganic salt, room temperature
Lower stirring 10 minutes.
Wherein, in reagent 3, the final concentration of each component or percentage composition are respectively as follows: heparin 0.5～3IU/ml, buffer agent 10～
50mM, inorganic salt 0.1～0.3M.
Preferably, preparation method may also include the step that the solution of reagent 1, reagent 2 or reagent 4 is made lyophilized preparation, tool
Body refers to the preparation technology of freeze dried powder of the prior art, to obtain the higher reagent of stability 1, reagent 2 or reagent 4.
Additionally, mentioned reagent 1, reagent 2, reagent 4 and reagent 3 are also separately added into the preservative of 0.2%, refer to existing
The method mentioned in technology adds, and does not repeats at this.
It is a further object of the present invention to provide the application of above-mentioned antithrombin activity detectable, will mentioned reagent conduct
Detectable is for preparing the detectable of detection antithrombin activity.
As a kind of preferred version, this detectable can prepare corresponding detection kit, including box body with equipped with above-mentioned
The reagent bottle of reagent, reagent bottle is positioned in described box body.
As a kind of preferred version, above-mentioned detection kit comprises reagent 1, reagent 2 respectively.Test sample is treated in the detection of this test kit
In product during antithrombin activity, being mixed with reagent 2 by testing sample and hatch, adding after reagent 1 mixes and hatch, reading is treated
The signal intensity of chromophoric substrate in test sample product；Finally by signal calculated intensity and the standard curve ratio of concentration known standard substance
To obtaining antithrombin activity in blood.
As a kind of preferred version, above-mentioned detection kit comprises reagent 1, reagent 4 or reagent 3 respectively.This test kit is examined
When surveying antithrombin activity in testing sample, testing sample mixed with reagent 3 and hatch, adding reagent 4 and mix and hatch,
Add after reagent 1 mixes and hatch, then read the signal intensity of chromophoric substrate in testing sample；Finally by signal calculated
Intensity and and the standard curve comparison of concentration known standard substance obtain antithrombin activity in blood.
It is a further object of the present invention to provide a kind of method detecting antithrombin activity, particular by chromophoric substrate
Method, after testing sample is mixed with above-mentioned antithrombin activity Activity determination reagent and hatched, the signal of detection chromophoric substrate is strong
Degree；And the absorbance signal produced is to be inversely proportional to the activity of antithrombase in sample.Finally by signal calculated intensity also
The activity of antithrombase in testing sample is obtained with standard curve comparison.
Above-mentioned detection method comprises the following steps:
Step a, the making of standard curve: calibration object and corresponding signal intensity thereof according to different antithrombin activities are painted
Standard curve processed；
Step b, testing sample processes: obtains the blood plasma of testing sample, mixes with above-mentioned antithrombin activity detectable
And hatch；
Step c, the signal intensity of chromophoric substrate in detection testing sample；
Step d, by calculating in testing sample, signal intensity is also and standard curve comparison obtains anticoagulation in testing sample
The activity of enzyme.
Preferably, step a is to use buffer agent to be diluted to by the human plasma calibration object containing antithrombase of concentration known
Four kinds or above concentration known, as standard solution；Further according to antithrombin activities different in human plasma calibration object with record
Corresponding light absorption value draw standard curve.
It is highly preferred that the blood plasma of testing sample is in the ratio of 9:1 and citric acid by the venous blood of fresh extraction in step b
It is centrifuged after anti-freezing liquid mixing, separates and i.e. obtain testing sample blood plasma.
It is highly preferred that the temperature that in step b, testing sample processes is 37 DEG C, incubation temperature is also 37 DEG C.
As a kind of preferred version, the concrete operations of step b are: by testing sample blood plasma and reagent 2 with the volume of 1:24
Ratio mixes at 37 DEG C and hatches 2 minutes, and (testing sample blood plasma reagent adding 2 with the volume ratio of reagent 1 is to be subsequently adding reagent 1
1:1), mix at 37 DEG C and hatch 2 minutes.
As another kind of preferred version, the concrete operations of step b are: by testing sample blood plasma and reagent 3 with the body of 1:24
Long-pending ratio mixes at 37 DEG C and hatches 2 minutes, is subsequently adding reagent 4 (testing sample blood plasma reagent adding 3 and the volume ratio of reagent 4
For 1:1), mix at 37 DEG C and hatch 2 minutes, being eventually adding reagent 1 (testing sample blood plasma reagent adding 3 reagent adding 4 and reagent
The volume ratio of 1 is 2:1), mix at 37 DEG C and hatch 2 minutes.
Preferably, the absorbance under above-mentioned signal is 405nm.
It is highly preferred that testing sample is put into detection absorbance in spectrophotometer by step c, it is no less than 5 standing time
Minute.
The detection method of above-mentioned antithrombin activity is applied on automatic tester (such as coaglation analyzer or biochemical analysis
Instrument) actual conditions that carries out detecting is:
Setup parameter on automonitor: reaction temperature: 37 DEG C；Detection wavelength: 405nm；Detection method: end-point method or
Dynamic method.
It should be noted that, when using different automatic tester (coaglation analyzer or biochemistry analyzer), specifically join
Number should adjust accordingly according to instrument difference.
Compared with prior art, the advantage of the present invention is:
The present invention is the Chromogenic assay based on FXa, it is to avoid liver in the Chromogenic assay based on thrombin
The impact that element cofactor II, hirudin, argatroban, dabigatran or other direct thrombin inhibitor bring.This
Bright detection stability and reproducible, has the highest sensitivity and accuracy, can accurate response antithrombin activity, obtain
Result accurately and reliably, and finds the abnormal patient of internal antithrombin activity, thus provides foundation for treatment.Additionally, the present invention
The FXa used is sufficiently stable, is extremely suitable for use in self-reacting device such as coaglation analyzer or biochemistry analyzer, thus realizes
Automatization, patient, without carrying out repeated detection, contributes to clinical application, has easy and simple to handle, and highly sensitive feature makes
Scope more extensive.Therefore, it has a extensive future.
Accompanying drawing explanation
Fig. 1 is the standard curve of antithrombin activity used by embodiment 1；
Fig. 2 is the standard curve of antithrombin activity used by embodiment 2；
Fig. 3 is the standard curve of antithrombin activity used by embodiment 3；
Fig. 4 is the standard curve of antithrombin activity used by embodiment 4.
Detailed description of the invention
The present invention is made the most in detail with embodiment and comparative example, intactly illustrates below in conjunction with the accompanying drawings.Used below
Reagent or equipment are commercially available kind, if no special instructions, operate the most to specifications, do not repeat at this.
It is below that the present invention is further illustrated in conjunction with specific embodiments, but is not construed as limitation of the invention.
1, experiment material and reagent
Tris (trishydroxymethylaminomethane) is biological super pure, purchased from U.S. Sigma-Aldrich (Sigma-
Aldrich, is called for short Sigma)；
Hydrochloric acid is super pure, purchased from Sigma；
Sodium chloride is biological super pure, purchased from Sigma；
PEG-8 000, standard level, purchased from Sigma；
The chromophoric substrate of FXa and FXa, purchased from Chromogenix；
Heparin, USP heparin sodium content measuring standard product, purchased from Beijing Ai Dehaoke International Technologies, INC.；
Hemoglobin, lyophilized powder, purchased from Sigma；
Bilirubin, dry powder, purchased from Sigma；
Triglyceride, standard level, purchased from Sigma；
Heparin co factor II, 50% glycerite, purchased from Sigma；
Hirudin, lyophilized powder, purchased from Sigma；
Argatroban, dry powder, purchased from Sigma；
Dabigatran, standard level, purchased from Sigma.
2, test apparatus
Coaglation analyzer is purchased from STAGO；
Blood plasma is from chain hospital.
Embodiment 1
The test kit one that the preparation present invention provides, comprises reagent 1, reagent 4 and reagent 3, is used for measuring anticoagulation in blood
The activity of enzyme.
Particularly as follows: reagent 1 is the chromophoric substrate of FXa, its concrete preparation method is: take CH3SO2-D-Leu-Gly-Arg-p-
Nitroanilide AcOH is dissolved in the Tris buffer that concentration is 30mM, adds salt acid for adjusting pH value to 7.4；Add chlorine
Changing sodium and PEG-8 000, prepare reagent 1 after stirring, its final concentration is respectively as follows: CH3SO2-D-Leu-Gly-Arg-p-
Nitroanilide AcOH 2mM, sodium chloride 0.2M, PEG-8 000 1%.
Reagent 4 is FXa, and its concrete preparation method is: takes FXa and is dissolved in the Tris buffer that concentration is 30mM, adds salt
Acid for adjusting pH value is to 7.4；Adding sodium chloride and PEG-8 000, prepare reagent 4 after stirring, its final concentration is respectively as follows:
FXa 0.5 μM, sodium chloride 0.2M, PEG-8 000 1%.
Reagent 3 is the diluent containing heparin, and its concrete preparation method is: taking heparin is dissolved in the Tris that concentration is 30mM and delays
Rush liquid, add salt acid for adjusting pH value to 7.4；Adding sodium chloride, prepare reagent 3 after stirring, its final concentration is respectively as follows: heparin
2IU/ml, sodium chloride 0.2M.
Standard curve making:
(1) take titer to mix homogeneously in the ratio of 1:24 with reagent 3, and hatch 2 minutes, hatch and the temperature reacted is equal
It it is 37 DEG C.
(2) then take 200 μ L said mixtures and add 200 μ L reagent 4, mix homogeneously, and hatch 2 minutes, incubation temperature
It it is 37 DEG C.
(3) it is subsequently adding 200 μ L reagent 1, mix homogeneously, is placed in spectrophotometer and measures extinction at 405 nano wave lengths
Spend 5 minutes.
(4) according to antithrombin activity titer gradient concentration and corresponding light absorption value, linear equation is used to draw standard
Curve, asks for an interview accompanying drawing 1, and its standard curve formula is y=-0.0105x+1.5587 (R2=0.9891).
Sample detection:
Take 3 parts of blood plasma mixed in 1:24 ratio to mix with reagent 3, hatch 2 minutes for 37 DEG C, then take 200 μ l mixing
Thing adds 200 μ l reagent 4,37 DEG C and hatches 2 minutes, is eventually adding 200 μ l reagent 1,37 DEG C reaction and measures suction at 405 nano wave lengths
Luminosity 5 minutes.Every part of sample repeated measure twice, log, signal calculated intensity, and standard curve comparison are resisted
The activity of thrombin.
The detection specificity of gained test kit one, sensitivity, the range of linearity and stability are shown in following experiment.
Embodiment 2
Under environment same as in Example 1, preparation is the present invention provide test kit two, comprises reagent 1 and reagent 2, is used for surveying
Determine the activity of antithrombase in blood.
Particularly as follows: reagent 1 is the chromophoric substrate of FXa, its concrete preparation method is: take Suc-Ile-Glu (γ-
Piperidyl)-Gly-Arg-p-nitroanilide HCl is dissolved in the Tris buffer that concentration is 30mM, adds hydrochloric acid and adjusts
Joint pH value is to 7.4；Adding sodium chloride and PEG-8 000, prepare reagent 1 after stirring, its final concentration is respectively as follows: Suc-
Ile-Glu (γ-Piperidyl)-Gly-Arg-p-nitroanilide HCl 2mM, sodium chloride 0.2M, Polyethylene Glycol-
8000 1%.
Reagent 2 is the diluent containing FXa and heparin, and its concrete preparation method is: take FXa successively and heparin is dissolved in concentration
For the Tris buffer of 30mM, add salt acid for adjusting pH value to 7.4；Add sodium chloride and PEG-8 000, make after stirring
Obtaining reagent 2, its final concentration is respectively as follows: FXa 0.5 μM, heparin 2IU/ml, sodium chloride 0.2M, PEG-8 0001%.
Standard curve making:
(1) take titer to mix homogeneously in the ratio of 1:24 with reagent 2, and hatch 2 minutes, hatch and the temperature reacted is equal
It it is 37 DEG C.
(2) then take 200 μ L said mixtures and add 200 μ L reagent 2, mix homogeneously, be placed in spectrophotometer 405
Nano wave length measures absorbance 5 minutes, and temperature is 37 DEG C.
(3) according to antithrombin activity titer gradient concentration and corresponding light absorption value, linear equation is used to draw standard
Curve, asks for an interview accompanying drawing 2, and its standard curve formula is y=-0.0096x+1.4425 (R2=0.9928).
Sample detection:
Take 3 parts of blood plasma mixed in 1:24 ratio to mix with reagent 2, hatch 2 minutes for 37 DEG C, then take 200 μ l mixing
Thing adds 200 μ l reagent 1,37 DEG C reaction and measures absorbance 5 minutes at 405 nano wave lengths.Every part of sample repeated measure twice, note
Record result of the test, signal calculated intensity, and standard curve comparison obtain the activity of antithrombase.
The detection specificity of gained test kit two, sensitivity, the range of linearity and stability are shown in following experiment.
Embodiment 3
With the difference of embodiment 1, the present embodiment is only that in reagent 1 that chromophoric substrate is CH3O-CO-D-CHA-Gly-Arg-
p-nitroanilide·AcOH.Other preparation methoies and detection operation are consistent with embodiment 1.
According to antithrombin activity titer gradient concentration and corresponding light absorption value, linear equation is used to draw standard bent
Line, asks for an interview accompanying drawing 3, and its standard curve formula is y=-0.0088x+1.3884 (R2=0.9898).
The detection specificity of gained test kit three, sensitivity, the range of linearity and stability are shown in following experiment.
Embodiment 4
With the difference of embodiment 2, the present embodiment is only that in reagent 1 that chromophoric substrate is 4-Mbs-D-Arg-Gly-Arg-p-
nitroanilide·2HCl.Other preparation methoies and detection operation are consistent with embodiment 2.
According to antithrombin activity titer gradient concentration and corresponding light absorption value, linear equation is used to draw standard bent
Line, asks for an interview accompanying drawing 4, and its standard curve formula is y=-0.0107x+1.6526 (R2=0.985).
The detection specificity of gained test kit four, sensitivity, the range of linearity and stability are shown in following experiment.
Embodiment 5
With the difference of embodiment 1, the present embodiment is only that in reagent 1 that chromophoric substrate is Benzoyl-Ile-Glu-Gly-
Arg-p-nitroanilide·HCl.Other preparation methoies and detection operation are consistent with embodiment 1.
The testing result of the specificity of this test kit, sensitivity, the range of linearity and stability is deposited with embodiment 1 the data obtained
Concordance in theoretical range of error.
Embodiment 6
With the difference of embodiment 2, the present embodiment is only that in reagent 1 that chromophoric substrate is N-α-Z-D-Arg-Gly-Arg-p-
nitroanilide·2HCl.Other preparation methoies and detection operation are consistent with embodiment 1.
The testing result of the specificity of this test kit, sensitivity, the range of linearity and stability is deposited with embodiment 2 the data obtained
Concordance in theoretical range of error.
Embodiment 7
With the difference of embodiment 1, the present embodiment is only that in reagent 1 that chromophoric substrate is Boc-D-Arg-Gly-Arg-p-
nitroanilide·2HCl.Other preparation methoies and detection operation are consistent with embodiment 1.
The testing result of the specificity of this test kit, sensitivity, the range of linearity and stability is deposited with embodiment 1 the data obtained
Concordance in theoretical range of error.
Embodiment 8
With the difference of embodiment 2, the present embodiment is only that in reagent 1 that chromophoric substrate is Acetyl-D-Arg-Gly-Arg-p-
nitroanilide·2HCl.Other preparation methoies and detection operation are consistent with embodiment 2.
The testing result of the specificity of this test kit, sensitivity, the range of linearity and stability is deposited with embodiment 2 the data obtained
Concordance in theoretical range of error.
Embodiment 9
With the difference of embodiment 1, the present embodiment is only that in reagent 1 that chromophoric substrate is 4-Nz-D-Arg-Gly-Arg-p-
nitroanilide·2HCl.Other preparation methoies and detection operation are consistent with embodiment 1.
The testing result of the specificity of this test kit, sensitivity, the range of linearity and stability is deposited with embodiment 1 the data obtained
Concordance in theoretical range of error.
Embodiment 10
With the difference of embodiment 2, the present embodiment is only that in reagent 1 that chromophoric substrate is CH3OCO-D-CHG-Gly-Arg-p-
nitroanilide·AcOH.Other preparation methoies and detection operation are consistent with embodiment 2.
The testing result of the specificity of this test kit, sensitivity, the range of linearity and stability is deposited with embodiment 2 the data obtained
Concordance in theoretical range of error.
Embodiment 11
The present embodiment is with the difference of embodiment 1:
In reagent 1, each component is final concentration of: chromophoric substrate 0.1mM, Tris buffer 50mM, sodium chloride 0.1M, PEG-
8000 0.5%；
In reagent 4, each component is final concentration of: FXa 0.05 μM, Tris buffer 50mM, sodium chloride 0.1M, PEG-8000
0.5%；
In reagent 3, each component is final concentration of: heparin 0.3U/mL, Tris buffer 50mM, sodium chloride 0.1M, PEG-8000
0.5%.Other preparation methoies and detection operation are consistent with embodiment 1.
The testing result of the specificity of this test kit, sensitivity, the range of linearity and stability is deposited with embodiment 1 the data obtained
Concordance in theoretical range of error.
Embodiment 12
The present embodiment is with the difference of embodiment 2:
In reagent 1, each component is final concentration of: chromophoric substrate 3mM, Tris buffer 50mM, sodium chloride 0.1M, PEG-8000
0.5%；
In reagent 2, each component is final concentration of: FXa 1.5 μMs, heparin 0.3U/mL, Tris buffer 50mM, sodium chloride
0.1M, PEG-8000 0.5%.Other preparation methoies and detection operation are consistent with embodiment 2.
The testing result of the specificity of this test kit, sensitivity, the range of linearity and stability is deposited with embodiment 2 the data obtained
Concordance in theoretical range of error.
Embodiment 13
The present embodiment is with the difference of embodiment 1:
In reagent 1, each component is final concentration of: chromophoric substrate 1mM, Tris buffer 50mM, sodium chloride 0.1M, PEG-8000
0.5%；
In reagent 4, each component is final concentration of: FXa 2 μMs, Tris buffer 50mM, sodium chloride 0.1M, PEG-8000
0.5%；
In reagent 3, each component is final concentration of: heparin 3U/mL, Tris buffer 50mM, sodium chloride 0.1M, PEG-8000
0.5%.Other preparation methoies and detection operation are consistent with embodiment 1.
The testing result of the specificity of this test kit, sensitivity, the range of linearity and stability is deposited with embodiment 1 the data obtained
Concordance in theoretical range of error.
Embodiment 14
The present embodiment is only that with the difference of embodiment 2:
In reagent 1, each component is final concentration of: chromophoric substrate 1mM, Tris buffer 50mM, sodium chloride 0.1M, PEG-8000
0.5%；
In reagent 2, each component is final concentration of: FXa 2 μMs, heparin 3U/mL, Tris buffer 50mM, sodium chloride 0.1M,
PEG-8000 0.5%.Other preparation methoies and detection operation are consistent with embodiment 2.
The testing result of the specificity of this test kit, sensitivity, the range of linearity and stability is deposited with embodiment 1 the data obtained
Concordance in theoretical range of error.
The detection of the specificity of test kit, sensitivity, the range of linearity and stability
1. test kit specific detection
Table 1 detects antithrombin activity and measures test kit specificity
Result shows: the specificity of the detection kit that the present invention provides is high, with heparin co factor II, hirudin, A Jia
Qu Ban, the multiple factor such as dabigatran is not intersected.
2. test kit sensitivity technique and the detection of the test kit range of linearity: absorbance detection under 405nm wavelength
Table 2 detects antithrombin activity and measures test kit sensitivity
Result shows: test kit of the present invention can detect that the antithrombin activity of 25%, and the detection sensitivity of the method is extremely
It is 25% less.Antithrombin activity is when 0%～125%, linearly >=0.98, and the scope 0%～125% of detection.
3. reagent stability detection
Table 3 detects the reagent stability that embodiment 1 prepares
Result shows: the antithrombin activity detectable that the present invention provides when room temperature 25 DEG C stably at least 5 days, 4
DEG C time stably at least 3 months.
From above-mentioned experimental result, the present invention utilizes Chromogenic assay to detect the activity of antithrombase, and detection is stable
Property and reproducible, has the highest sensitivity and accuracy.Additionally, the present invention is also used in self-reacting device such as blood coagulation analysis
On instrument or biochemistry analyzer, it is achieved automatization contributes to clinical application.
Finally it is necessary described herein: above example is served only for making technical scheme the most in detail
Ground explanation, it is impossible to being interpreted as limiting the scope of the invention, those skilled in the art is according to the foregoing of the present invention
The nonessential improvement of some made and adjustment belong to protection scope of the present invention.
Claims (10)
1. antithrombin activity detectable, including reagent 1 and reagent 2, it is characterised in that: reagent 1 is the chromophoric substrate of FXa,
Reagent 2 is the diluent containing FXa and heparin.
2. antithrombin activity detectable, including reagent 1, reagent 4 and reagent 3, it is characterised in that: reagent 1 is the color development of FXa
Substrate, reagent 4 is FXa, and reagent 3 is the diluent containing heparin.
Antithrombin activity detectable the most according to claim 1 and 2, it is characterised in that: the chromophoric substrate of described FXa
Any in following substrate:
CH3SO2-D-Leu-Gly-Arg-p-nitroanilide·AcOH；
CH3OCO-D-CHG-Gly-Arg-p-nitroanilide·AcOH；
CH 3OCO-D-CHA-Gly-Arg-p-nitroanilide·AcOH；
Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide·HCl；
Suc-Ile-Glu(γ-Piperidyl)-Gly-Arg-p-nitroanilide·HCl；
N-α-Z-D-Arg-Gly-Arg-p-nitroanilide·2HCl；
Boc-D-Arg-Gly-Arg-p-nitroanilide·2HCl；
Acetyl-D-Arg-Gly-Arg-p-nitroanilide·2HCl；
4-Nz-D-Arg-Gly-Arg-p-nitroanilide·2HCl；
4-Mbs-D-Arg-Gly-Arg-p-nitroanilide·2HCl。
4. preparation antithrombin activity detectable method described in claim 1 or 2, it is characterised in that: include preparing respectively
Reagent 1, reagent 2, reagent 4 or reagent 3.
Antithrombin activity detectable the most according to claim 13, it is characterised in that the step preparing reagent 1 is:
Chromophoric substrate is dissolved in buffer agent；Adding hydrochloric acid, regulation pH value is to 7～8；Sequentially add inorganic salt and surface activity
Agent, stirs 10 minutes under room temperature.
Antithrombin activity detectable the most according to claim 13, it is characterised in that the step preparing reagent 2 is:
Successively FXa and heparin are dissolved in buffer agent；Adding hydrochloric acid, regulation pH value is to 7～8；Sequentially add inorganic salt and surface activity
Agent, stirs 10 minutes under room temperature.
Antithrombin activity detectable the most according to claim 13, it is characterised in that the step preparing reagent 4 is:
FXa is dissolved in buffer agent；Adding hydrochloric acid, regulation pH value is to 7～8；Sequentially add and stir under inorganic salt and surfactant, room temperature
Mix 10 minutes.
Antithrombin activity detectable the most according to claim 13, it is characterised in that the step preparing reagent 3 is:
Heparin is dissolved in buffer agent；Regulation pH value is to 7～8；Add inorganic salt, stir 10 minutes under room temperature.
The application of antithrombin activity detectable the most according to claim 3, it is characterised in that: described reagent is as inspection
Test agent is for preparing the detectable of detection antithrombin activity.
10. the method detecting antithrombin activity, it is characterised in that detection method comprises the following steps:
Step a, the making of standard curve: calibration object and corresponding signal intensity thereof according to different antithrombin activities draw mark
Directrix curve；
Step b, testing sample processes: obtains the blood plasma of testing sample, mixes with above-mentioned antithrombin activity detectable and incubate
Educate；
Step c, the signal intensity of chromophoric substrate in detection testing sample；
Step d, by calculating in testing sample, signal intensity is also and standard curve comparison obtains antithrombase in testing sample
Activity.
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| CN107091814A (en) * | 2017-06-23 | 2017-08-25 | 宁波艾科生物科技有限公司 | A kind of detection reagent of liquid instant blood heparin concentration |
| CN107153043A (en) * | 2017-06-23 | 2017-09-12 | 宁波艾科生物科技有限公司 | A kind of liquid instant Antiprothrombin antibodies determine reagent |
| CN107167439A (en) * | 2017-06-30 | 2017-09-15 | 上海贞元诊断用品科技有限公司 | A kind of kit that dabigatran content is detected based on Chromogenic assay |
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| CN107727587A (en) * | 2017-09-22 | 2018-02-23 | 宁波瑞源生物科技有限公司 | A kind of Antithrombin III assay kit and its detection method |
| CN110423795A (en) * | 2019-08-02 | 2019-11-08 | 大连工业大学 | A kind of measuring method of thrombin-inhibiting activity |
| CN117030672A (en) * | 2023-08-11 | 2023-11-10 | 中国药科大学 | Fluorescent probe for detecting activity of coagulation factor FXIa inhibitor in complex system and application thereof |
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| CN110423795A (en) * | 2019-08-02 | 2019-11-08 | 大连工业大学 | A kind of measuring method of thrombin-inhibiting activity |
| CN117030672A (en) * | 2023-08-11 | 2023-11-10 | 中国药科大学 | Fluorescent probe for detecting activity of coagulation factor FXIa inhibitor in complex system and application thereof |
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