CN106884035A - A kind of factor X activator titration method - Google Patents
A kind of factor X activator titration method Download PDFInfo
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- CN106884035A CN106884035A CN201510940717.9A CN201510940717A CN106884035A CN 106884035 A CN106884035 A CN 106884035A CN 201510940717 A CN201510940717 A CN 201510940717A CN 106884035 A CN106884035 A CN 106884035A
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Abstract
The invention provides a kind of assay method of factor X activator potency, the method for the present invention calculates the activity of the A of F Ⅹ according to the amount of the material of generation ρ NA in the unit interval.The method of the present invention does not use the A of F Ⅹ and a standard items of F Ⅹ to calculate product to be tested potency indirectly, reduce error in test process, accurately, convenient, fast, the method for the present invention has preferable repeatability, precision, durability and linear, the potency of measure F Ⅹ A that can stablize.
Description
Technical field
The invention belongs to biological products, Pharmaceutical Analysis technical field, more particularly to a kind of factor X activator
Titration method.
Background technology
Containing various enzymes that can influence mammal blood coagulation system in snake venom, these enzymes are cascaded with clotting mechanism and put
There is the strict selectivity of comparing to act between each clotting factor in big reaction, wherein factor X activator is a kind of energy
The enzyme with proteolytic enzyme characteristic of factor X is enough directly acted on, in snakes such as Viperinae, Crotalinae, Elapidaes
It is widely distributed in poison.
The hematological system of human normal is in the dynamic equilibrium promoted between solidifying-anti-freezing, when body is subject to external source to injure or disease
During disease interference, the coagulation homeostasis of body are broken, and start the reaction of blood coagulation waterfall Cascaded amplification to resist the influence of foeign element.So
It is finally factor X regardless of whether being external source coagulation pathway or endogenous coagulation pathway（Coagulation Factor
Ⅹ, F Ⅹ）The state of activation is activated as, that is, the factor X for activating（Coagulation Factor Ⅹ Activated, F
Ⅹa）, a of F Ⅹ activation factor generation fibrin ferments, performance hemoglutination.My company separates from Brazilian spearhead viper venom
To factor X activator（The A of Coagulation Factor Ⅹ Activator, F Ⅹ）, it is that molecular weight is 79000Da
One-component glycoprotein, containing a heavy chain and two light chains, with calcium ion dependence.In vitro study shows, in Ca2+
The A of F Ⅹ can generate a of F Ⅹ with specific hydrolysis F Ⅹ in the presence of under conditions of, closely make one the-citric acid clotting of plasma.In vivo studies
Research shows that the A of F Ⅹ can substantially shorten the docking bleeding time of normal mouse, with preferable anastalsis, in view of the A of F Ⅹ are highly
Specific effect feature, tentatively judges also to have preferable haemostatic effect to haemophilia A mouse, can alleviate clinic at present and only lean on
Supplement of coagulation factors VIII treats the situation of haemophilia A, the financial burden of patient can be reduced, with preferable pharmacoeconomics
Value.
At present, the activity determination method of the A of F Ⅹ can be divided into two major classes by the difference according to substrate specificity:First major class is day
Right substrate method, i.e., with human plasma as substrate, the time solidified with the addition blood plasma of the A of F Ⅹ carries out the determination of activity of the A of F Ⅹ,
If notification number is the Chinese patent application of CN101104847B.The method is widely used in early 1980s, and method is normal
Rule, but need to estimate judgement PCT, the subjective interference of examined person, and different manufacturers people's Quality Control blood plasma and ordinary people
The content difference of citric acid plasma F Ⅹ is larger, and the contents of F Ⅹ are most important to the A determinations of activity of F Ⅹ in blood plasma, therefore causes the A of F Ⅹ to be imitated
Valency test result is inconsistent.Second major class is made a living color substrate method, i.e., hydrolyzing chromogenic substrate by a of F Ⅹ has characteristic absorption
The determination of activity that chromogenic product is carried out, can be divided into two step method and the A external standard methods of F Ⅹ again.Two step method is to act on Ⅹ 1 sections of F in the A of F Ⅹ
Terminator is added after time, while adding chromogenic substrate, the growing amount according to a of F in special time Ⅹ calculates the activity of the A of F Ⅹ.This
Method needs to introduce a standard items of F Ⅹ, it is impossible to exclude that may be present between the early stage time of activation and a growing amounts of F Ⅹ
Non-linear relation（Li Jing, Liang Chenggang, poplarization are new etc., the continuous rate determination round spot adder factor X activator of chromogenic substrate
Activity [J] Chinese Journal of New Drugs, 2007,16（18）：1419）, thus can not accurately reflect that the active function of the A of F Ⅹ is special
Point.The A external standard methods of F Ⅹ（Such as the Chinese patent application of Publication No. CN102798598）By detecting the A standard items various concentrations of F Ⅹ
The absorbance of ρ NA at 405nm, draws standard curve and calculates testing sample potency.The method need to provide the A standard items of F Ⅹ, and F
Ⅹ A standardizations are demarcated by substrate visual method of blood plasma again, and the problem of first kind method mentioned above equally occurs.
The content of the invention
It is an object of the invention to provide a kind of titration method of factor X activator, the method be with blood coagulation because
Son Ⅹ（FⅩ）It is reaction substrate, the factor X of the A of the F Ⅹ activation generation activation of F Ⅹ（FⅩa）, a of F Ⅹ hydrolysis chromogenic substrate lifes
Into the paranitroanilinum of displaing yellow（ρNA）, there is absorption maximum in 405nm wavelength, under conditions of F Ⅹ and chromogenic substrate are enough, ρ
NA concentration is active related to the A of F Ⅹ and has linear dependence in certain scope, according to generation ρ NA in the unit interval
The amount of material calculates the activity of the A of F Ⅹ.Titration method of the invention can accurately determine the A potency of F Ⅹ, without the A of F Ⅹ and F
Ⅹ a standard items calculate product to be tested potency indirectly, reduce error in test process, accurate, convenient, fast.The method has preferable
Repeatability, precision, durability and linear, the potency of measure F Ⅹ A that can stablize.
To achieve the above object, the present invention provides following technical scheme：
A kind of titration method of factor X activator, methods described includes：
1）Factor X is diluted with cushioning liquid A, the concentration for obtaining Stuart factor is the solution of 1.0U/ml~2.0U/ml
1；
2）Factor X activator is diluted with cushioning liquid B, solution 2 is made；
3）By chromogenic substrate deionized water dissolving, the solution 3 that concentration is 1.5mg/ml~3.0mg/ml is obtained；
4）Solution 1, solution 2, cushioning liquid C are mixed, is put in 37 DEG C of water-baths and is incubated 8 ~ 12 minutes, add cushioning liquid D and molten
Liquid 3, is mixed, and the 0th, 1,2,3 minutes absorbance values are determined under 405nm wavelength, and solution 2 is replaced with cushioning liquid B, is operated with method,
As blank；The volume ratio of the solution 1, solution 2, solution 3, cushioning liquid C and buffering solution D is 1: 1: 1: 1: 1；
5）Paranitroanilinum concentration is calculated by computing formula, factor X activator potency is obtained；
Wherein, the cushioning liquid A is with Na+Ion concentration meter concentration delays-biphosphate for the disodium hydrogen phosphate of 0.02mol/L
Sodium fliud flushing, the tris-HCI buffer with trishydroxymethylaminomethane content meter concentration as 0.02mol/L or
With Na+Ion concentration meter concentration is the sodium citrate-citric acid buffer solution of 0.02mol/L, containing 0.1mol/L sodium chloride and
0.001mol/L benzamidine hcls, pH is 7.4；
The cushioning liquid B is with Na+Ion concentration meter concentration is the disodium hydrogen phosphate-sodium dihydrogen phosphate buffering of 0.01mol/L
Liquid, with trishydroxymethylaminomethane content meter concentration as 0.01mol/L tris-HCI buffers or with Na+
Ion concentration meter concentration is 0.01mol/L sodium citrate-citric acid buffer solutions, and pH is 8.0；
The cushioning liquid C is with Na+Ion concentration meter concentration is the disodium hydrogen phosphate-sodium dihydrogen phosphate buffering of 0.1mol/L
Liquid, the tris-HCI buffer with trishydroxymethylaminomethane content meter concentration as 0.1mol/L or with Na+
Ion concentration meter concentration for 0.1mol/L sodium citrate-citric acid buffer solution, containing 0.3mol/L sodium chloride,
0.002mol/L benzamidine hcls and 0.01mol/L calcium chloride, pH is 7.5；
The cushioning liquid D is with Na+Ion concentration meter concentration for 1mol/L disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution,
Tris-HCI buffer with trishydroxymethylaminomethane content meter concentration as 1mol/L or with Na+Ion
Content meter concentration is the sodium citrate-citric acid buffer solution of 1mol/L, contains 0.75mol/L sodium chloride, 0.05mol/L
EDTA-Na2, pH is 8.3；
The concentration of the solution 2 control can make generation ρ NA concentration be 0.05 μm of olml-1·min-1~0.25 μm of olml-1·min-1；
Described computing formula is
△ASampleRepresent t minutes of sample and the difference of the absorption value of the 0th minute；△AIt is emptyRepresent t minutes of blank product
With the difference of the absorption value of the 0th minute；T represents the minute of the sample and blank in terms of min, t ﹥ 0, it is preferable that t=
1st, 2 or 3；VtRepresent the reaction cumulative volume in terms of ml；10.4 represent with ml μm of ol-1·cm-1The molar absorptivity system of the ρ NA of meter
Number；1 represents the light path in terms of cm；VsIt is the A test sample volumes of F Ⅹ in terms of ml.
In an embodiment of the invention, the concentration of the solution 1 is 1.0U/ml~1.5U/ml；Preferably
1.25U/ml。
In an embodiment of the invention, the concentration of the solution 3 is 1.5mg/ml~3.0mg/ml；It is preferred that
It is 1.5mg/ml.
In an embodiment of the invention, the factor X behaviour source or ox source clotting factor.
In an embodiment of the invention, the chromogenic substrate is to contain p-nitrophenyl amine groups（ρNA）'s
Small peptide or its hydrochloride.
In an embodiment of the invention, the factor X activator comes from snake venom.
In an embodiment of the invention, the snake venom be selected from spearhead viper venom, cyclopentadienyl viper venom and
One or more in Vipera russelli venom.
The present inventor has found under study for action, under conditions of factor X and chromogenic substrate are enough, paranitroanilinum
Amount of the growing amount only with factor X activator is relevant, and with preferable linear dependence, coefficient R >=0.99.Should
Preferably, RSD is 2.1% to the repeatability of method.Intermediate precision and durability are preferable, including different tests tester experiment RSD is
2.8%th, different manufacturers chromogenic substrate test RSD is that 2.2%, different manufacturers factor X test RSD is purple 6.8%, different manufacturers
Outer spectrophotometer test RSD is 2.6%.The rate of recovery of the method is 100.2%.The method can accurately, stably as can be seen here
Test out the potency of factor X activator, hence it is evident that better than blood plasma substrate method and F Ⅹ a, F Ⅹ A standard items external standard method, can
Accurate Clinical practice dosage is provided, the security and validity of clinical application is improved.
Brief description of the drawings
Fig. 1 is the A extension rates of the F Ⅹ linear relationship curve with ρ NA concentration reciprocal during the A of F Ⅹ determine for examination concentration.
Fig. 2 is the linear relationship curve for calculating the rate of recovery.
Specific embodiment
The present invention is further illustrated below by specific embodiment, it should be understood, however, that, the only conduct of these embodiments
Specifically describe in more detail and be used, and be not to be construed as limiting the present invention in any form.
Embodiment 1Factor X（FⅩ）Determine with chromogenic substrate preferred concentration
The selection people of F Ⅹ source, chromogenic substrate selection S-2765.By the pH7.4 0.02mol/L Tris-HCl buffer solutions of F Ⅹ（Contain
0.1mol/L NaCl, 0.01mol/L benzamidine hcls）0.75U/ml, 1.0U/ml, 1.25U/ml, 1.5U/ are configured to respectively
The solution of ml, 2.0U/ml, as solution 1.Take the A test sample pH8.0 0.01mol/L Tris-HCl buffer solutions of F Ⅹ moderately dilute
Release, as solution 2.Chromogenic substrate S-2765 is taken, it is 1.0mg/ml, 1.5mg/ to be diluted to deionized water dissolving and respectively concentration
Ml, 2.0mg/ml, the solution of 2.5 mg/ml, 3.0mg/ml, as solution 3.PH7.5 0.1mol/L Tris-HCl are prepared to delay
Fliud flushing（0.3mol/L containing NaCl, benzamidine hcl 0.002mol/L, CaCl20.01mol/L）As cushioning liquid C；Prepare
PH8.3 1mol/L Tris-HCl buffer solutions（0.75mol/L containing NaCl, EDTA-Na20.05mol/L）As cushioning liquid
D；PH8.0 0.01mol/L Tris-HCl buffer solutions replace the A test samples of F Ⅹ as blank.Open uv-spectrophotometric
Meter, treats that temperature reaches 37 DEG C, and solution 1,2, each 0.2ml additions cuvettes of buffer solution C are taken respectively, mixes, in uv-spectrophotometric
The online insulation 10min of 37 ± 0.5 DEG C of meter, adds 0.2ml cushioning liquid D and 0.2ml solution 3, mixes, and continues to be incubated, detection
The absorption value of the 0th, 1,2,3 minutes under 405nm wavelength.Data are substituted into formula, the concentration of ρ NA is calculated.F Ⅹ and S-2765 are preferred
Concentration results such as table 1 below.
The preferred concentration result of the F Ⅹ and S-2765 of table 1.
Result shows that in the range of the U/ml of 1.0U/ml~2.0, S-2765 concentration is in 1.5mg/ for the concentration of F Ⅹ in the present invention
In the range of ml~3.0mg/ml, the concentration mensuration result of ρ NA is relatively stable, and F Ⅹ and S-2765 are enough.
Embodiment 2Determinations of the A of F Ⅹ for examination concentration
By F Ⅹ（People source）With pH7.4 0.02mol/L Tris-HCl buffer solutions（NaCl containing 0.1mol/L, 0.01mol/L hydrochloric acid
Benzenecarboximidamide）Buffer into 1.25 U/ml solution, as solution 1；Take the A test sample pH8.0 0.01mol/L of F Ⅹ
Tris-HCl buffer solutions dilute 20000 times, 12500 times, 8333 times, 5000 times, 4000 times, 3030 times respectively, used as solution 2；
Chromogenic substrate S-2765 is taken, with deionized water dissolving and the solution that concentration is 1.5mg/ml is diluted to, as solution 3；Prepare
PH7.5 0.2mol/L Tris-HCl buffer solutions（0.6mol/L containing NaCl, benzamidine hcl 0.004mol/L, CaCl2
0.02mol/L）As cushioning liquid C；Prepare pH8.3 1mol/L Tris-HCl buffer solutions（0.75mol/L containing NaCl,
EDTA-Na20.05mol/L）As cushioning liquid D；PH8.0 0.01mol/L Tris-HCl buffer solutions replace the A test samples of F Ⅹ
As blank.Ultraviolet specrophotometer is opened, treats that temperature reaches 37 DEG C, solution 1,2, each 0.2ml of cushioning liquid C are taken respectively
Add cuvette, mix, in the online insulation 10min of 37 ± 0.5 DEG C of ultraviolet specrophotometer, add 0.2ml cushioning liquid D and
0.2ml solution 3, mixes, and continues to be incubated, the absorption value of the 0th, 1,2,3 minutes under detection 405nm wavelength.Data are substituted into formula,
Calculate the concentration of ρ NA.The A potency of F Ⅹ（U/ml）Concentration × the extension rate of=ρ NA.
Paranitroanilinum（ρNA）Concentration range determine result as shown in table 2, best effort concentration that is near and limiting the A of F Ⅹ
Scope.
The A concentration of 2 F of table Ⅹ determines result.
With the inverse of test sample extension rate as abscissa, ρ NA concentration is ordinate, draws curve（See Fig. 1）, linear side
Journey is Y=856.61X+0.00162, R2=0.99118.Can be drawn by the data of table 2, the A of F Ⅹ are diluted into suitable concentration in the present invention
Make the ρ NA concentration of generation in 0.042~0.274 μm of olml-1·min-1In the range of, the titer plateaus of the A of F Ⅹ.In the present invention
In, the A test samples of acceptable diluent F Ⅹ make the ρ NA concentration of generation in 0.05 μm of olml-1·min-1~0.25 μm of olml-1·
min-1In the range of.
Embodiment 33 crowdes of F Ⅹ A titrations
By F Ⅹ（People source）With pH7.4 0.02mol/L Tris-HCl buffer solutions（NaCl containing 0.1mol/L, 0.01mol/L hydrochloric acid
Benzenecarboximidamide）Buffer into 1.25 U/ml solution, as solution 1；Take 3 crowdes of A test sample pH8.0 0.01mol/L of F Ⅹ
Tris-HCl buffer solutions moderately dilute, and can make the concentration of ρ NA in the range of 0.05 μm of ol/ml~0.25 μm ol/ml, as
Solution 2；Chromogenic substrate S-2765 is taken, with deionized water dissolving and the solution that concentration is 1.5mg/ml is diluted to, as solution 3；
Prepare pH7.5 0.2mol/L Tris-HCl buffer solutions（0.6mol/L containing NaCl, benzamidine hcl 0.004mol/L, CaCl2
0.02mol/L）As cushioning liquid C；Prepare pH8.3 1mol/L Tris-HCl buffer solutions（0.75mol/L containing NaCl,
EDTA-Na20.05mol/L）As cushioning liquid D；PH8.0 0.01mol/L Tris-HCl buffer solutions replace the A of F Ⅹ for examination
Product are used as blank.Ultraviolet specrophotometer is opened, treats that temperature reaches 37 DEG C.Take respectively solution 1,2, cushioning liquid C it is each
0.2ml adds cuvette, mixes, and in 37 ± 0.5 DEG C of online insulation 10min of ultraviolet specrophotometer, adds 0.2ml cushioning liquid
D and 0.2ml solution 3, mixes, and continues to be incubated, the absorption value of the 0th, 1,2,3 minutes under detection 405nm wavelength.Data are substituted into public
Formula calculates the concentration of ρ NA.Formula is as follows：
Potency, V are calculated so that lot number is the A of 150107F Ⅹ as an exampletRepresent reaction cumulative volume 1ml, VsRepresent the A test sample volumes of F Ⅹ
0.2ml, t represent minute 3min, the △ A of sample and blank productSampleRepresent the sample absorption with the 0th minute in the 3rd minute
The difference 1.092 of value, △ AIt is emptyRepresent the difference 0.000 of blank position 3 minutes and the absorption value of the 0th minute, substitution formula
The concentration for calculating ρ NA is 0.175 μm of olml-1·min-1, the A extension rates of 150107 batch F Ⅹ are 5000, draw effect
Valency is 875U/ml, and protein content is 0.155mg/ml, and Rate activity is 875/0.155=5645U/mg.
The A unit of activity of F Ⅹ definition in the present invention：At 37 DEG C, under the conditions of pH 8.0,1 μm of ol p-nitrophenyl of generation in 1min
Enzyme amount required for amine, is defined as 1 unit.
3 batch factor X activator potency are determined by the method for embodiment 3, as a result such as table 3.
33 batches of titration results of the A of F Ⅹ of table.
Embodiment 4The A titrations methods of F Ⅹ repeatability
The A potency of F Ⅹ is determined by the method for embodiment 3, test sample 150107 is taken and is tested, be repeated 6 times, record data, as a result such as table 4.
The A titrations methods of 4 F of table Ⅹ repeatability result.
Embodiment 5The A titration methods Intermediate precisions of F Ⅹ are investigated
The A potency of F Ⅹ is determined by the method for embodiment 3, test sample 150107 is taken, different personnel's different times are tested, record is surveyed
Examination data, as a result such as table 5.
The A titration method Intermediate precision results of 5 F of table Ⅹ.
Embodiment 6The A titration methods durabilities of F Ⅹ are investigated
1）Different manufacturers chromogenic substrate S-2765 determines the A titer plateaus of F Ⅹ
Determined by the method for embodiment 3, take test sample 150107, potency is determined with different manufacturers chromogenic substrate S-2765, record is surveyed
Examination data, as a result such as table 6.
The different manufacturers S-2765 of table 6 determines the A potency results of F Ⅹ.
2）Different manufacturers reaction substrate F Ⅹ determines the stability of the A potency of F Ⅹ
The A potency of F Ⅹ is determined by the method for embodiment 3, test sample 150107 is taken, is determined with different manufacturers reaction substrate F Ⅹ, record is surveyed
Examination data, as a result such as table 7.
7 different manufacturers F of table Ⅹ determines the A potency results of F Ⅹ.
3）Different model ultraviolet specrophotometer determines the stability of the A potency of F Ⅹ
The A potency of F Ⅹ is determined by the method for embodiment 3, test sample 150107 is taken, is determined with different model ultraviolet specrophotometer and imitated
Valency, records test data, as a result such as table 8.
The A potency results of 8 different model Instrument measuring F of table Ⅹ.
Embodiment 7The linear and degree of accuracy of the A titration methods of F Ⅹ（The rate of recovery）
By the method for embodiment 3 determine the A potency of F Ⅹ, take test sample 150107, respectively dilute 8333 times, 7143 times, 6250 times,
5000 times, 4000 times, test data is recorded, as a result such as table 9.
Table 9 is linear and rate of recovery result.
With the inverse of test sample extension rate as abscissa, ρ NA concentration is ordinate, draws curve（See Fig. 2）, linear side
Journey is y=848.13x+0.00282 R2=0.99356, it is linear good.
Bring extension rate inverse into standard curve, calculate theoretical potency, survey potency with theoretical potency ratio to reclaim
Rate, rate of recovery average value is 100.2%, RSD=2.6%, and the inventive method degree of accuracy is good.
Embodiment 8The A titrations methods of F Ⅹ and direct blood plasma are the comparing of substrate method measurement result in the present invention
1）Common human plasma is the potency that direct substrate method determines the A of F Ⅹ
The A test samples of F Ⅹ are taken, lot number 150107 dilutes 800 times, as confession with the 0.01mol/L Tris-HCl buffer solutions of pH8.0
Test sample solution.Take common human plasma（Originate blood station）, 37 DEG C of water-baths are melted completely, standby.It is molten calcium chloride to be prepared with deionized water
Liquid, concentration is 0.02mol/L, standby.
Take in the common human plasmas of 0.2ml to small test tube in 37 examination preheating 2min, add 0.1ml need testing solutions, add
0.1ml 0.02mol/L calcium chloride solutions, shake up, timing, and the time of white floc sedimentation, measurement result such as table 10 occurs in record blood plasma
It is shown.
By table 10 below as can be seen that test result differs greatly between blood plasma, it is impossible to which accurate stable tests out the A potency of F Ⅹ.
The common human plasma of table 10 is that direct substrate determines the A potency results of F Ⅹ.
2）People's blood coagulation Quality Control blood plasma is the potency that direct substrate method determines the A of F Ⅹ
The A test samples of F Ⅹ are taken, lot number 150107 dilutes 800 times, as confession with the 0.01mol/L Tris-HCl buffer solutions of pH8.0
Test sample solution.Take people's blood coagulation Quality Control blood plasma（Liang Ge producers are purchased from respectively）, each producer takes 2 batches, molten with deionized water 1ml respectively
Solution, it is standby.Calcium chloride solution is prepared with deionized water, concentration is 0.02mol/L, standby.
Take in 0.2ml people's blood coagulation Quality Control blood plasma to small test tube in 37 to preheating 2min, add 0.1ml need testing solutions, plus
Enter 0.1ml 0.02mol/L calcium chloride solutions, shake up, timing, the time of white floc sedimentation, measurement result such as table occurs in record blood plasma
Shown in 11.
By table 11 below as can be seen that test result has differences between same manufacturer's different batches blood plasma, different factories
Blood plasma test result between family there is also difference, it is impossible to which accurate stable tests out the A potency of F Ⅹ.
The people's blood coagulation Quality Control blood plasma of table 11 is that direct substrate determines the A potency results of F Ⅹ.
Although present invention has been a certain degree of description, it will be apparent that, do not departing from the spirit and scope of the present invention
Under the conditions of, the appropriate change of each condition can be carried out.It is appreciated that the invention is not restricted to the embodiment, and it is attributed to right
It is required that scope, its equivalent for including each factor.
Claims (10)
1. a kind of factor X activator titration method, it is characterised in that：Methods described includes：
1）Factor X is diluted with cushioning liquid A, the concentration for obtaining Stuart factor is the solution of 1.0U/ml~2.0U/ml
1；
2）Factor X activator is diluted with cushioning liquid B, solution 2 is made；
3）By chromogenic substrate deionized water dissolving, the solution 3 that concentration is 1.5mg/ml~3.0mg/ml is obtained；
4）Solution 1, solution 2, cushioning liquid C are mixed, is put in 37 DEG C of water-baths and is incubated 8 ~ 12 minutes, add cushioning liquid D and molten
Liquid 3, is mixed, and the 0th, 1,2,3 minutes absorbance values are determined under 405nm wavelength, and solution 2 is replaced with cushioning liquid B, is operated with method,
As blank；The volume ratio of the solution 1, solution 2, solution 3, cushioning liquid C and buffering solution D is 1: 1: 1: 1: 1；
5）Paranitroanilinum concentration is calculated by computing formula, factor X activator potency is obtained；
Wherein, the cushioning liquid A is with Na+Ion concentration meter concentration delays-biphosphate for the disodium hydrogen phosphate of 0.02mol/L
Sodium fliud flushing, the tris-HCI buffer with trishydroxymethylaminomethane content meter concentration as 0.02mol/L or
With Na+Ion concentration meter concentration is the sodium citrate-citric acid buffer solution of 0.02mol/L, containing 0.1mol/L sodium chloride and
0.001mol/L benzamidine hcls, pH is 7.4；
The cushioning liquid B is with Na+Ion concentration meter concentration is the disodium hydrogen phosphate-sodium dihydrogen phosphate buffering of 0.01mol/L
Liquid, with trishydroxymethylaminomethane content meter concentration as 0.01mol/L tris-HCI buffers or with Na+
Ion concentration meter concentration is 0.01mol/L sodium citrate-citric acid buffer solutions, and pH is 8.0；
The cushioning liquid C is with Na+Ion concentration meter concentration for 0.1mol/L disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution,
Tris-HCI buffer with trishydroxymethylaminomethane content meter concentration as 0.1mol/L or with Na+From
Sub- content meter concentration is the sodium citrate-citric acid buffer solution of 0.1mol/L, contains 0.3mol/L sodium chloride, 0.002mol/L
Benzamidine hcl and 0.01mol/L calcium chloride, pH is 7.5；
The cushioning liquid D is with Na+Ion concentration meter concentration for 1mol/L disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, with
Trishydroxymethylaminomethane content meter concentration is for the tris-HCI buffer of 1mol/L or with Na+Ion contains
Gauge concentration is the sodium citrate-citric acid buffer solution of 1mol/L, contains 0.75mol/L sodium chloride, 0.05mol/L EDTA-
Na2, pH is 8.3；
The concentration of the solution 2 control can make generation ρ NA concentration be 0.05 μm of olml-1·min-1~0.25 μm of olml-1·min-1；
Described computing formula is
；
△ASampleT minutes is represented with the 0th minute difference of sample absorption value；△AIt is emptyT minutes is represented with the 0th minute blank
The difference of product absorption value；T represents the minute of the sample and blank in terms of min, t ﹥ 0；VtRepresent the reaction in terms of ml
Cumulative volume；10.4 represent with ml μm of ol-1·cm-1The molar absorption coefficient of the ρ NA of meter；1 represents the light path in terms of cm；VsBe with
The A test sample volumes of F Ⅹ of ml meters.
2. the method for claim 1, it is characterised in that the concentration of the solution 1 is 1.0U/ml~1.5U/ml.
3. method as claimed in claim 1 or 2, it is characterised in that the concentration of the solution 1 is 1.25U/ml.
4. the method as any one of claim 1 ~ 3, it is characterised in that the concentration of the solution 3 be 1.5mg/ml~
3.0mg/ml。
5. the method as any one of claim 1 ~ 4, it is characterised in that the concentration of the solution 3 is 1.5mg/ml.
6. the method as any one of claim 1 ~ 5, it is characterised in that t=1,2 or 3 in the formula.
7. the method as any one of claim 1 ~ 6, it is characterised in that the factor X behaviour source or Niu Yuan.
8. the method as any one of claim 1 ~ 7, it is characterised in that the chromogenic substrate is to contain paranitroanilinum
Group（ρNA）Small peptide or its hydrochloride.
9. the method as any one of claim 1 ~ 8, it is characterised in that the factor X activator comes from snake
Poison.
10. method as claimed in claim 9, it is characterised in that the snake venom is selected from spearhead viper venom, cyclopentadienyl viper venom
With one or more in Vipera russelli venom.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114250265A (en) * | 2021-12-15 | 2022-03-29 | 深圳传世生物医疗有限公司 | Liquid type factor X determination kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1282793A (en) * | 1999-07-28 | 2001-02-07 | 武汉埃克玛生物技术有限公司 | Reagent for testing angiotonin converzyme and its application and test method |
| CN102798598A (en) * | 2011-05-25 | 2012-11-28 | 兆科药业（合肥）有限公司 | Method for detecting enzymatic activity of phospholipid-depending factor X activator |
-
2015
- 2015-12-16 CN CN201510940717.9A patent/CN106884035B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1282793A (en) * | 1999-07-28 | 2001-02-07 | 武汉埃克玛生物技术有限公司 | Reagent for testing angiotonin converzyme and its application and test method |
| CN102798598A (en) * | 2011-05-25 | 2012-11-28 | 兆科药业（合肥）有限公司 | Method for detecting enzymatic activity of phospholipid-depending factor X activator |
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| 李晶等: "生色底物连续速率法测定圆斑蝰蛇凝血因子X激活剂的活性", 《中国新药杂志》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114250265A (en) * | 2021-12-15 | 2022-03-29 | 深圳传世生物医疗有限公司 | Liquid type factor X determination kit |
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