CN108508220A - A kind of PT reagents are for measuring the active purposes of FXa in blood plasma - Google Patents
A kind of PT reagents are for measuring the active purposes of FXa in blood plasma Download PDFInfo
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Abstract
The present invention provides a kind of PT reagents for measuring the active purposes of FXa in blood plasma, and the active methods of FXa in blood plasma are measured using PT reagents, the PT reagent safeties are easy to get, instead of expensive rare RVV X, the FXa activity determination methods are simple and easy to operate, efficient and sensible, can FXa activity in effecting reaction blood plasma.
Description
Technical field
The invention belongs to pharmaceutical technology field, a kind of PT reagents are related generally to for measuring the active purposes of FXa in blood plasma,
The active methods of FXa in human or animal's blood plasma are measured more particularly to using PT reagents.
Background technology
In coagulation cascade reaction, coagulation factor 10 (FX) plays key effect, either endogenous or exogenous
Coagulation pathways, finally all act on FX, and activation FX generates active FXa.Under the catalytic action of FXa, factor generates
Fibrin ferment plays hemoglutination.Therefore, the active variations of FXa play key effect, by right in the reaction of blood coagulation waterfall
The active detections of FXa, the case where can largely reacting blood coagulation function.
In addition, with the development of anticoagulant, oral FXa inhibition gradually replaces traditional anticoagulant heparin, low molecular weight
Heparin and warfarin become the mainstream medicine of anticoagulation in the market.Oral FXa inhibitor (such as razaxaban, Eliquis, according to
Du Shaban etc.), compared with traditional anticoagulant, better efficacy uses more convenient, patient medication compliance higher.Nevertheless,
Oral FXa inhibits similar with traditional anticoagulant, excessive or in special circumstances in dosage, can cause different degrees of
Hemorrhage side effect.If bleeding part is in brain or important organ, this side effect may be fatal.Ordinary circumstance
Under, plasma prothrombin time (PT) and partial prothrombin time (APTT) can react the coagulation function feelings of patient's body
Condition.For taking the patient of oral FXa inhibitor, blood concentration and the extension degree correlation of PT or APTT are poor, are taking
In the case of taking orally FXa inhibitor with recommended dose, often only part extends or does not extend by PT or APTT.Therefore, lead to
The active in body of oral FXa inhibitor can not be reflected well by crossing measurement patients blood plasma PT or APTT.The study found that in blood plasma
FXa maximum inhibitions and the blood concentration of oral FXa inhibitor are proportionate, can be more solidifying than tradition by the active detections of FXa
Blood index PT and APTT preferably reflect patient in body coagulation function.According to patient FXa activity, judge patient whether anti-freezing
Degree or anti-freezing are insufficient, and then adjust drug dosage so that the use of exploitation FXa inhibitor is more safe and effective.
CN101535498A discloses a kind of method and apparatus that electrochemical process measures Xa factor inhibitor in blood sample,
Testing element based on dry chemistry and test element analysis system shorten test period, reduce difficulty of test, but need Ruse
Your adder is malicious, FX in blood plasma is converted to active FXa first, but RVV-X yield is extremely low, and it is inconvenient to preserve, expensive, and
Use certain risk.
Therefore it provides a kind of using easy, safe and accurate, have suitable for the active detection methods of FXa in the case of various
Significance and bright prospects.
Invention content
In view of the deficiencies of the prior art and actual demand, a kind of PT reagents of present invention offer are used to measure FXa in blood plasma
Active purposes, the PT reagent safeties are easy to get, instead of expensive rare RVV-X, the easy easily behaviour of the FXa activity determination methods
Make, efficient and sensible, FXa activity in energy effecting reaction blood plasma.
For this purpose, the present invention uses following technical scheme：
In a first aspect, a kind of PT reagents of present invention offer are for measuring the active purposes of FXa in blood plasma.
In the present invention, replace the active RVV-X of conventional detection FXa, RVV-X in the prior art that can disobey with PT reagents
Rely exogenous and intrinsic coagulation access, is directly FXa by FX catalysis, and then Coagulation test occurs in the presence of calcium ion, and
Entirely different with it using the PT reagents detection active testing principles of FXa in the application, blood is after internal take out, using anti-
Solidifying agent sodium citrate is combined with calcium ion, that is, factor IV so that has lacked thromboplastin and IV in blood plasma, PT examinations are added
Blood coagulation waterfall can be activated to react after agent, under the collective effect of proconvertin, form answering for thromboplastin, IV and VII
Object is closed, in the presence of calcium ion, Stuart factor generates active FXa, and FXa forms compound with factor IV, Va,
It is catalyzed prothrombin and generates FIIa, i.e. fibrin ferment, in the presence of athrombia inhibitor, fibrin ferment will be catalyzed coagulation factor
I (fibrinogen) generates fibrin, and fibrin be combined with each other so that therefore the clotting of plasma in the reaction system must
A certain amount of thrombin inhibitor is added.
Preferably, the PT reagents include Medulla Leporis seu Oryctolagi extract, glucose, albumin, potassium sorbate and calcium chloride.
Preferably, the mass concentration of the Medulla Leporis seu Oryctolagi extract is 20-50mg/mL, such as can be 20mg/mL, 22mg/
mL、23mg/mL、25mg/mL、28mg/mL、30mg/mL、32mg/mL、35mg/mL、38mg/mL、40mg/mL、42mg/mL、
45mg/mL, 48mg/mL or 50mg/mL, preferably 30-45mg/mL.
Preferably, the mass concentration of the glucose be 15-35mg/mL, such as can be 15mg/mL, 16mg/mL,
18mg/mL、20mg/mL、22mg/mL、23mg/mL、25mg/mL、26mg/mL、28mg/mL、30mg/mL、32mg/mL、34mg/
ML or 35mg/mL, preferably 20-30mg/mL.
Preferably, the mass concentration of the albumin is 80-120mg/L, such as can be 80mg/L, 82mg/L, 85mg/
L、86mg/L、88mg/L、90mg/L、92mg/L、95mg/L、96mg/L、98mg/L、100mg/L、102mg/L、103mg/L、
105mg/L, 108mg/L, 110mg/L, 112mg/L, 113mg/L, 115mg/L, 116mg/L, 118mg/L or 120mg/L, preferably
For 90-110mg/L.
Preferably, the mass concentration of the potassium sorbate be 450-550mg/L, such as can be 450mg/L, 452mg/L,
455mg/L、458mg/L、460mg/L、465mg/L、470mg/L、475mg/L、480mg/L、485mg/L、490mg/L、
495mg/L、500mg/L、505mg/L、510mg/L、515mg/L、520mg/L、525mg/L、530mg/L、535mg/L、
540mg/L, 545mg/L or 550mg/L, preferably 480-520mg/L.
Preferably, the molar concentration of the calcium chloride be 0.01-0.1mol/L, such as can be 0.01mol/L,
0.012mol/L、0.0.125mol/L、0.0128mol/L、0.013mol/L、0.014mol/L、0.015mol/L、
0.016mol/L、0.018mol/L、0.02mol/L、0.023mol/L、0.025mol/L、0.028mol/L、0.03mol/L、
0.032mol/L、0.035mol/L、0.038mol/L、0.04mol/L、0.042mol/L、0.043mol/L、0.045mol/L、
0.048mol/L、0.05mol/L、0.052mol/L、0.055mol/L、0.056mol/L、0.058mol/L、0.06mol/L、
0.062mol/L、0.065mol/L、0.068mol/L、0.07mol/L、0.08mol/L、0.09mol/L、0.095mol/L、
0.098mol/L or 0.1mol/L, preferably 0.012-0.05mol/L.
Preferably, the mass concentration of the PT reagents is 7.5-30%, for example, can be 7.5%, 8.0%, 8.5%,
9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%,
20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%, 29.5%
Or 30.0%, preferably 10-25%.
Second aspect, the present invention provide a kind of active methods of FXa in measurement blood plasma, include the following steps：
(1) blood sample is acquired, anti-coagulants is added and carries out anti-freezing, centrifuging and taking blood plasma；
(2) thrombin inhibitor is added in the blood plasma into step (1), adds PT reagents, be incubated；
(3) FXa chromophoric substrates are added into the blood plasma after incubation, are detected.
Preferably, the anti-coagulants is sodium citrate.
Preferably, the mass concentration of the anti-coagulants is 2-8%, for example, can be 2%, 2.5%, 3%, 3.5%, 4%,
4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5% or 8%, preferably 3-5%.
Preferably, the volume ratio of the anti-coagulants and the blood sample is 1:(5-15), such as can be 1:5、1:6、1:7、
1:8、1:9、1:10、1:11、1:12、1:13、1:14 or 1:15, preferably 1:(8-12).
Preferably, the rotating speed of the step (1) centrifugation be 2000-5000rpm, such as can be 2000rpm,
2200rpm、2500rpm、2800rpm、3000rpm、3300rpm、3500rpm、3800rpm、4000rpm、4200rpm、
4500rpm, 4800rpm or 5000rpm, preferably 2500-4000rpm.
Preferably, the centrifugation time be 5-15min, such as can be 5min, 6min, 7min, 8min, 9min,
10min, 11min, 12min, 13min, 14min or 15min, preferably 8-12min.
Preferably, the thrombin inhibitor is hirudin.
In the present invention, the effect using thrombin inhibitor be FXa generate after catalysis factor generate it is active
Fibrin ferment, fibrin ferment can generate fibrin with catalysis fibre proteinogen, so that the clotting of plasma, hinders entire reaction system
Liquid condition, be added thrombin inhibitor after so that fibrin ferment can not play a role, and prevent the solidification of blood plasma.
Preferably, the mass concentration of the thrombin inhibitor be 0.5-3mg/mL, such as can be 0.5mg/mL,
0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1.0mg/mL、1.1mg/mL、1.2mg/mL、1.3mg/mL、
1.4mg/mL、1.5mg/mL、1.6mg/mL、1.7mg/mL、1.8mg/mL、1.9mg/mL、2.0mg/mL、2.1mg/mL、
2.2mg/mL, 2.3mg/mL, 2.4mg/mL, 2.5mg/mL, 2.6mg/mL, 2.7mg/mL, 2.8mg/mL, 2.9mg/mL or
3.0mg/mL, preferably 0.8-2mg/mL.
Preferably, the mass concentration of the PT reagents is 7.5-30%, for example, can be 7.5%, 8.0%, 8.5%,
9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%,
20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%, 29.5%
Or 30.0%, preferably 10-25%.
Preferably, the temperature of the incubation is 20-40 DEG C, for example, can be 20 DEG C, 21 DEG C, 22 DEG C, 23 DEG C, 24 DEG C, 25
DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 30 DEG C, 31 DEG C, 32 DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C or 40 DEG C, it is excellent
It is selected as 25-37 DEG C.
Preferably, the time of the incubation be 1-10min, such as can be 1min, 2min, 3min, 4min, 5min,
6min, 7min, 8min, 9min or 10min, preferably 3-8min.
Preferably, the FXa chromophoric substrates are S-2765.
Preferably, the molar concentration of the FXa chromophoric substrates is 300-500 μM, for example, can be 300 μM, 310 μM, 320
μM, 330 μM, 350 μM, 360 μM, 370 μM, 380 μM, 400 μM, 410 μM, 420 μM, 440 μM, 450 μM, 480 μM or 500 μM,
Preferably 350-450 μM.
Preferably, the detection is detected using microplate reader.
Preferably, the wavelength of the detection be 400-420nm, such as can be 400nm, 401nm, 402nm, 403nm,
404nm、405nm、406nm、407nm、408nm、409nm、410nm、411nm、412nm、413nm、414nm、415nm、
416nm, 417nm, 418nm, 419n or 420nm.
Preferably, the detection specifically comprises the following steps：Since reacting and originating, absorbance is measured every 1-3min,
Obtained absorbance is subjected to linear fit, calculates FXa activity values.
In the present invention, the specific method for calculating FXa activity values is as follows：
Using absorbance value when 3min subtract reaction starting when absorbance value, then divided by reaction time 3min to get FXa
Activity, with FXa activity datas before administration be 100%, the FXa activity datas at each time point compare therewith to get FXa after administration
Inhibiting rate.
Preferably, described method includes following steps：
(1) blood sample is acquired, it is 1 to be added with blood sample volume ratio:The mass concentration of (5-15) is that the anti-coagulants of 2-8% is resisted
Solidifying, 2000-5000rpm centrifugations 5-15min takes blood plasma；
(2) hirudin being added in the blood plasma into step (1), adds PT reagents, 20-40 DEG C is incubated 1-10min,
In, the mass concentration of hirudin is 0.5-3mg/mL, and the mass concentration of PT reagents is 7.5-30%；
(3) S-2765 is added into the blood plasma after incubation, the molar concentration of the S-2765 is 300-500 μM, in wavelength
It is detected for 400-420nm, since reacting and originating, absorbance is measured every 1-3min, by obtained absorbance into line
Property fitting, calculate FXa activity values.
Compared with prior art, the present invention has the advantages that：
(1) present invention discover that can be used for detecting FXa activity using PT reagents, to replace expensive and few yield RVV-X,
Detection sensitivity is high, as a result accurately and reliably, greatly reduces use cost and risk；
(2) method provided by the invention is overcome the limitation of conventional method, is suitable for each using easy, safe and accurate
FXa Activity determinations in the case of kind, including isolated experiment and experiments in vivo are detectable, and do not limit blood plasma kind, are one
Kind wide spectrum, easy FXa assay methods.
Description of the drawings
Fig. 1 is that dog blood concentration and FXa inhibiting rates relational graph at any time is administered orally in razaxaban in embodiment 1.
Specific implementation mode
Further to illustrate the present invention technological means and its effect taken, below by way of specific implementation mode come into
One step illustrates technical scheme of the present invention, but the present invention is not limited in scope of embodiments.
Material and instrument
1, drug and reagent：
Razaxaban, Tianjin Inst. of Materia Medica Co., Ltd provide, white powder, lot number 1603012；
S-2765, the production of Beijing Ai Dehaoke International Technologies, INC., lot number：170306；
FXa, the production of Beijing Ai Dehaoke International Technologies, INC., lot number：150625；
Lepirudin 023 ludon is provided by Institute of Radiation Medicine of Military Medical Science Institute, lot number 20140326；
Prothrombin time (Prothrombin Time, PT) assay kit, the production of virtue Pacific Ocean Science and Technology Ltd.
Product, lot number：011611A；
Wherein PT reagents：A) Medulla Leporis seu Oryctolagi extract (tissue thromboplastin)：40mg/mL；B) glucose：25mg/mL；C) white egg
In vain：100mg/L；D) potassium sorbate：500mg/L；E) calcium chloride：1/80mol/L.
2, animal：
Beagle dogs, half male and half female, weight 10-12kg, Shenyang Kangping institute of lab animals provide, animal productiong license
Card SCXK (the Liao Dynasty) 2014-0003.
3, instrument：
Rayto RT-6100 microplate reader, Rayto Life and Analytical Sciences Co., Ltd.'s product；
LD5-2A centrifuges, Beijing Medical Centrifugal Machine Factory's product.
Embodiment 1
1, animal administration and collection of specimens
Healthy adult Beagle dogs 6, half male and half female.The equal gavage of every dog gives the razaxaban of 1mg/kg, and solvent is
PEG400：Ethyl alcohol：Water=3：1：1, concentration is 0.5mg/mL, and administered volume is 2mL/kg.Before administration and after administration
Sodium citrate anti-freezing, 3000rpm centrifugations is respectively adopted in 0.5h, 1h, 2h, 3h, 4h, 6h, 9h, 12h, 15h, for 24 hours femoral vein blood
Blood plasma, packing is taken to be placed in -80 DEG C of refrigerators and preserve.
2, Indexs measure
Razaxaban content is measured using LC-MS/MS methods in blood plasma：50 μ are added in 50 μ L dogs plasma containing drugs (anticoagulant heparin)
L internal standards razaxaban (500ng/mL) methanol working solution and 100 μ L methanol solutions, vortex 1min, 4 DEG C of 12000rpm centrifugations
10min.Take supernatant in interpolation pipe, 4 DEG C of 12000rpm centrifuge 10min, and 5 μ L of sample introduction carry out LC-MS/MS quantitative analyses, obtain
Obtain the concentration data of original shape drug razaxaban in each plasma sample.
3, FXa determinations of activity
(1) blood sample is acquired, it is 1 to be added with blood sample volume ratio:The sodium citrate that 5 mass concentration is 2% carries out anti-freezing,
5000rpm centrifugations 5min takes blood plasma；
(2) hirudin is added in the blood plasma into step (1), adds PT reagents, 20 DEG C of incubation 10min, wherein leech
The mass concentration of element is 0.5mg/mL, and the mass concentration of PT reagents is 30%；
(3) S-2765 is added into the blood plasma after incubation, the molar concentration of the S-2765 is 300 μM, is in wavelength
400nm is detected, and since reacting and originating, absorbance is measured every 3min, and obtained absorbance is carried out linear fit, meter
Calculate FXa activity values；
Specific FXa activity value calculating methods are as follows：Absorbance value when reaction starting is subtracted using absorbance value when 3min,
Again divided by reaction time 3min to get FXa activity, with FXa activity datas before administration be 100%, each time point after administration
To get FXa inhibiting rates, as a result as shown in figure 1 and table 1 FXa activity datas compare therewith.
Table 1
It can be seen that after razaxaban 1mg/kg gavages give beagle dogs from table 1 and Fig. 1, blood concentration rises rapidly,
1h reaches Cmax after to administration, and later over time, blood concentration continuously decreases, until after administration for 24 hours, profit cuts down sand
Class is most of to be metabolized, and blood concentration is very low, consistent with blood concentration, and FXa inhibiting rates rise rapidly upon administration, 0.5h
When have reached 92.3%, until when 1h, FXa inhibiting rates reach maximum value 93.0%, later with the reduction of blood concentration, FXa suppressions
Rate processed continuously decreases, until administration after for 24 hours when, FXa inhibiting rates are only 0.2%.
Embodiment 2
Animal administration, collection of specimens, index detection method and FXa inhibiting rates computational methods are the same as embodiment 1.
FXa activity determination methods, include the following steps：
(1) blood sample is acquired, it is 1 to be added with blood sample volume ratio:The sodium citrate that 15 mass concentration is 8% carries out anti-freezing,
2000rpm centrifugations 15min takes blood plasma；
(2) hirudin is added in the blood plasma into step (1), adds PT reagents, 40 DEG C of incubation 1min, wherein leech
The mass concentration of element is 3mg/mL, and the mass concentration of PT reagents is 7.5%；
(3) S-2765 is added into the blood plasma after incubation, the molar concentration of the S-2765 is 500 μM, is in wavelength
420nm is detected, and since reacting and originating, absorbance is measured every 3min, and obtained absorbance is carried out linear fit, meter
FXa activity values are calculated, the results are shown in Table 2.
Table 2
From table 2 it can be seen that after razaxaban 1mg/kg gavages give beagle dogs, blood concentration rises rapidly, until giving
1h reaches Cmax after medicine, and later over time, blood concentration continuously decreases, until after administration for 24 hours, razaxaban is big
Part has been metabolized, and blood concentration is very low, consistent with blood concentration, and FXa inhibiting rates rise rapidly upon administration, when 0.5h
Reach 91.5%, until when 1h, FXa inhibiting rates reach maximum value 93.5%, later with the reduction of blood concentration, FXa inhibiting rates
Continuously decrease, until administration after for 24 hours when, FXa inhibiting rates are only 0.3%.
Embodiment 3
Animal administration, collection of specimens, index detection method and FXa inhibiting rates computational methods are the same as embodiment 1.
FXa activity determination methods, include the following steps：
(1) blood sample is acquired, it is 1 to be added with blood sample volume ratio:The sodium citrate that 9 mass concentration is 3.8% carries out anti-freezing,
2500rpm centrifugations 10min takes blood plasma；
(2) hirudin is added in the blood plasma into step (1), adds PT reagents, 25 DEG C of incubation 8min, wherein leech
The mass concentration of element is 1.5mg/mL, and the mass concentration of PT reagents is 20%；
(3) S-2765 is added into the blood plasma after incubation, the molar concentration of the S-2765 is 400 μM, is in wavelength
410nm is detected, and since reacting and originating, absorbance is measured every 2min, and obtained absorbance is carried out linear fit, meter
FXa activity values are calculated, the results are shown in Table 3.
Table 3
From table 3 it can be seen that after razaxaban 1mg/kg gavages give beagle dogs, blood concentration rises rapidly, until giving
1h reaches Cmax after medicine, and later over time, blood concentration continuously decreases, until after administration for 24 hours, razaxaban is big
Part has been metabolized, and blood concentration is very low, consistent with blood concentration, and FXa inhibiting rates rise rapidly upon administration, when 0.5h
Reach 92.8%, until when 1h, FXa inhibiting rates reach maximum value 94.1%, later with the reduction of blood concentration, FXa inhibiting rates
Continuously decrease, until administration after for 24 hours when, FXa inhibiting rates are only 0.2%.
Embodiment 4
Animal administration, collection of specimens, index detection method and FXa inhibiting rates computational methods are the same as embodiment 1.
FXa activity determination methods, include the following steps：
(1) blood sample is acquired, it is 1 to be added with blood sample volume ratio:The sodium citrate that 9 mass concentration is 3.8% carries out anti-freezing,
4000rpm centrifugations 8min takes blood plasma；
(2) hirudin is added in the blood plasma into step (1), adds PT reagents, 30 DEG C of incubation 5min, wherein leech
The mass concentration of element is 2mg/mL, and the mass concentration of PT reagents is 15%；
(3) S-2765 is added into the blood plasma after incubation, the molar concentration of the S-2765 is 450 μM, is in wavelength
415nm is detected, and since reacting and originating, absorbance is measured every 1min, and obtained absorbance is carried out linear fit, meter
FXa activity values are calculated, the results are shown in Table 4.
Table 4
From table 4, it can be seen that after razaxaban 1mg/kg gavages give beagle dogs, blood concentration rises rapidly, until giving
1h reaches Cmax after medicine, and later over time, blood concentration continuously decreases, until after administration for 24 hours, razaxaban is big
Part has been metabolized, and blood concentration is very low, consistent with blood concentration, and FXa inhibiting rates rise rapidly upon administration, when 0.5h
Reach 92.8%, until when 1h, FXa inhibiting rates reach maximum value 94.3%, later with the reduction of blood concentration, FXa inhibiting rates
Continuously decrease, until administration after for 24 hours when, FXa inhibiting rates are only 0.2%.
Embodiment 5
Animal administration, collection of specimens, index detection method and FXa inhibiting rates computational methods are the same as embodiment 1.
FXa activity determination methods, include the following steps：
(1) blood sample is acquired, it is 1 to be added with blood sample volume ratio:The sodium citrate that 9 mass concentration is 3.8% carries out anti-freezing,
3000rpm centrifugations 12min takes blood plasma；
(2) hirudin is added in the blood plasma into step (1), adds PT reagents, 37 DEG C of incubation 3min, wherein leech
The mass concentration of element is 0.8mg/mL, and the mass concentration of PT reagents is 25%；
(3) S-2765 is added into the blood plasma after incubation, the molar concentration of the S-2765 is 350 μM, is in wavelength
405nm is detected, and since reacting and originating, absorbance is measured every 3min, and obtained absorbance is carried out linear fit, meter
FXa activity values are calculated, the results are shown in Table 5.
Table 5
As can be seen from Table 5, after razaxaban 1mg/kg gavages give beagle dogs, blood concentration rises rapidly, until giving
1h reaches Cmax after medicine, and later over time, blood concentration continuously decreases, until after administration for 24 hours, razaxaban is big
Part has been metabolized, and blood concentration is very low, consistent with blood concentration, and FXa inhibiting rates rise rapidly upon administration, when 0.5h
Reach 92.2%, until when 1h, FXa inhibiting rates reach maximum value 92.9%, later with the reduction of blood concentration, FXa inhibiting rates
Continuously decrease, until administration after for 24 hours when, FXa inhibiting rates are only 0.2%.
By table 1-5 it is found that the active methods of detection FXa provided by the invention are simple and effective, embodiment 1-5 data stabilizations,
Collimation is good, and under each component concentration proportioning of the present invention and experiment condition, the FXa that can be quickly detected in blood plasma lives
Property, accuracy is high, genuine and believable for the pharmacodynamics and pharmacokinetics of evaluating drug.
To sum up, FXa detection methods provided by the invention replace the RVV-X of expensive rareness, drop with PT reagents cheap and easy to get
Experiment safety is greatly increased while low experimental cost, is laid the foundation for FXa correlative studys, and there is broad prospect of application.
Applicant states that the present invention illustrates the method detailed of the present invention, but the present invention not office by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, the selection etc. of concrete mode, all fall within protection scope of the present invention and the open scope.
Claims (10)
1. a kind of PT reagents are for measuring the active purposes of FXa in blood plasma.
2. purposes according to claim 1, which is characterized in that the PT reagents include Medulla Leporis seu Oryctolagi extract, glucose, white egg
In vain, potassium sorbate and calcium chloride；
Preferably, the mass concentration of the Medulla Leporis seu Oryctolagi extract is 20-50mg/mL, preferably 30-45mg/mL；
Preferably, the mass concentration of the glucose is 15-35mg/mL, preferably 20-30mg/mL；
Preferably, the mass concentration of the albumin is 80-120mg/L, preferably 90-110mg/L；
Preferably, the mass concentration of the potassium sorbate is 450-550mg/L, preferably 480-520mg/L；
Preferably, the molar concentration of the calcium chloride is 0.01-0.1mol/L, preferably 0.012-0.05mol/L.
3. purposes according to claim 1, which is characterized in that the mass concentration of the PT reagents is 7.5-30%, preferably
For 10-25%.
4. a kind of active methods of FXa in measurement blood plasma, which is characterized in that include the following steps：
(1) blood sample is acquired, anti-coagulants is added and carries out anti-freezing, centrifuging and taking blood plasma；
(2) thrombin inhibitor is added in the blood plasma into step (1), adds PT reagents, be incubated；
(3) FXa chromophoric substrates are added into the blood plasma after incubation, are detected.
5. according to the method described in claim 4, it is characterized in that, the anti-coagulants is sodium citrate；
Preferably, the mass concentration of the anti-coagulants is 2-8%, preferably 3-5%；
Preferably, the volume ratio of the anti-coagulants and the blood sample is 1:(5-15), preferably 1:(8-12).
6. method according to claim 4 or 5, which is characterized in that the rotating speed of step (1) described centrifugation is 2000-
5000rpm, preferably 2500-4000rpm；
Preferably, the time of the centrifugation is 5-15min, preferably 8-12min.
7. according to the method described in any one of claim 4-6, which is characterized in that the thrombin inhibitor is hirudin；
Preferably, the mass concentration of the thrombin inhibitor is 0.5-3mg/mL, preferably 0.8-2mg/mL.
8. according to the method described in any one of claim 4-7, which is characterized in that the mass concentration of the PT reagents is 7.5-
30%, preferably 10-25%；
Preferably, the temperature of the incubation is 20-40 DEG C, preferably 25-37 DEG C；
Preferably, the time of the incubation is 1-10min, preferably 3-8min；
Preferably, the FXa chromophoric substrates are S-2765；
Preferably, the molar concentration of the FXa chromophoric substrates is 300-500 μM, preferably 350-450 μM.
9. according to the method described in any one of claim 4-8, which is characterized in that the detection is examined using microplate reader
It surveys；
Preferably, the wavelength of the detection is 400-420nm；
Preferably, the detection specifically comprises the following steps：Since reacting and originating, absorbance is measured every 1-3min, will
The absorbance arrived carries out linear fit, calculates FXa activity values.
10. according to the method described in any one of claim 4-8, which is characterized in that include the following steps：
(1) blood sample is acquired, it is 1 to be added with blood sample volume ratio:The anti-coagulants that the mass concentration of (5-15) is 2-8% carries out anti-freezing,
2000-5000rpm centrifugations 5-15min takes blood plasma；
(2) hirudin is added in the blood plasma into step (1), adds PT reagents, 20-40 DEG C of incubation 1-10min, wherein water
The mass concentration of leech element is 0.5-3mg/mL, and the mass concentration of PT reagents is 7.5-30%；
(3) S-2765 is added into the blood plasma after incubation, the molar concentration of the S-2765 is 300-500 μM, is in wavelength
400-420nm is detected, and since reacting and originating, absorbance is measured every 1-3min, obtained absorbance is carried out linear
Fitting calculates FXa activity values.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110514851A (en) * | 2019-08-28 | 2019-11-29 | 深圳麦科田生物医疗技术有限公司 | The detection method and detection kit of blood platelet inhibiting rate |
| CN111856043A (en) * | 2020-08-28 | 2020-10-30 | 保定天岳生物工程有限公司 | Prothrombin time detection kit |
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| CN105264385A (en) * | 2013-06-28 | 2016-01-20 | 豪夫迈·罗氏有限公司 | Means and methods for universal calibration of anti-factor Xa tests |
| CN106591267A (en) * | 2017-01-04 | 2017-04-26 | 三诺生物传感股份有限公司 | Thromboplastin, extraction method thereof and PT reagent |
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2018
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| CN105264385A (en) * | 2013-06-28 | 2016-01-20 | 豪夫迈·罗氏有限公司 | Means and methods for universal calibration of anti-factor Xa tests |
| CN106591267A (en) * | 2017-01-04 | 2017-04-26 | 三诺生物传感股份有限公司 | Thromboplastin, extraction method thereof and PT reagent |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110514851A (en) * | 2019-08-28 | 2019-11-29 | 深圳麦科田生物医疗技术有限公司 | The detection method and detection kit of blood platelet inhibiting rate |
| CN111856043A (en) * | 2020-08-28 | 2020-10-30 | 保定天岳生物工程有限公司 | Prothrombin time detection kit |
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