CN106884035B - Method for measuring potency of blood coagulation factor X activator - Google Patents
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Abstract
The present invention provides a method for measuring the potency of a factor X activator, which calculates the activity of FXA based on the amount of a substance that produces rho NA per unit time. The method does not use the F XA and F Xa standard substances to indirectly calculate the potency of the product to be tested, reduces the error in the testing process, and is accurate, convenient and quick.
Description
Technical Field
The invention belongs to the technical field of biological product and drug analysis, and particularly relates to a method for measuring the potency of a blood coagulation factor X activator.
Background
The snake venom contains a plurality of enzymes which can influence the blood coagulation system of mammals, and the enzymes have strict specificity with each blood coagulation factor in the cascade amplification reaction of the blood coagulation mechanism, wherein the blood coagulation factor X activator is an enzyme which can directly act on the blood coagulation factor X and has the characteristic of proteolytic enzyme, and is widely distributed in snake venom of Viperidae, Viper subfamily, Elapidae, and the like.
The normal blood system of human body is in dynamic balance between procoagulant and anticoagulative state, and when the human body is in a state of being in contact with the anticoagulative stateWhen the blood coagulation cascade amplification reaction is interfered by exogenous injury or diseases, the blood coagulation balance of the organism is broken, and the blood coagulation cascade amplification reaction is started to resist the influence of external factors. However, both the extrinsic and intrinsic Coagulation pathways are eventually Activated to an Activated state, i.e., Activated Factor X (Activated Factor X Activated, F Xa), F Xa, activates prothrombin to generate thrombin, and thus, Coagulation is performed. A Coagulation Factor X Activator (F X A) is separated from Brazilian spearhead viper venom, is a glycoprotein with a single component and a molecular weight of 79000Da, contains a heavy chain and two light chains, and has calcium ion dependence. In vitro studies have shown that Ca is present2+In the presence of a catalyst capable of specifically hydrolyzing F X to form F Xa and thereby coagulating human-citric acid plasma. In vivo experimental research shows that FXA can obviously shorten the tail-breaking bleeding time of normal mice and has better hemostatic effect, and in view of the characteristic of high specificity of FXA, the FXA also has better hemostatic effect on hemophilia A mice by preliminary judgment, can relieve the situation that hemophilia A is treated only by supplementing blood coagulation factor VIII clinically at present, can reduce the economic burden of patients and has better pharmaceutical economic value.
At present, the method for measuring the activity of FXA can be divided into two major classes according to the difference of acting substrates, wherein the first major class is a natural substrate method, namely, the method for measuring the activity of FXA is carried out by taking human plasma as a substrate and adding the FXA into the plasma for coagulation, such as Chinese patent application with the publication number of CN 101104847B. The method is generally applied in the early 80 s of the 20 th century, is conventional, but needs visual inspection to judge the plasma coagulation time, is subject to subjective interference of testers, has larger difference between human quality control plasma of different manufacturers and common human citric acid plasma FX content, and the FX content in the plasma is important for F XA activity determination, so that F XA titer test results are inconsistent. The second main category of chromogenic substrate methods, i.e., activity assays by hydrolyzing chromogenic substrates with FXa to produce chromogenic products with characteristic absorptions, are classified into two-step methods and FXA external standard methods. The two-step method is to add a terminator after F Xa acts on F Xa for a certain period of time, and to add a chromogenic substrate, and to calculate the F Xa activity from the amount of F Xa produced in a specific period of time. The method needs to introduce an FXa standard substance, and cannot exclude the possible nonlinear relationship between the early stage time of activation and the generation amount of the FXa (plum crystal, Liangbangang, Yanghuaxin and the like, and the activity of the Vipera viper coagulation factor X activator is measured by the continuous rate of chromogenic substrates [ J ]. China New medicine journal, 2007,16 (18): 1419), so that the activity characteristics of the FXa cannot be accurately reflected. The FXA external standard method (such as Chinese patent application with the publication number CN 102798598) is used for calculating the titer of a sample to be detected by detecting the absorbance of different concentrations of the FXA standard substance at a rho NA position of 405nm and drawing a standard curve. This method requires the provision of the FXA standard, which is calibrated by a visual inspection method using plasma as a substrate, and also suffers from the problems of the first-mentioned method.
Disclosure of Invention
The invention aims to provide a method for measuring the titer of a factor X activator, which takes the Factor X (FX) as a reaction substrate, the FXA activates the FX to generate the activated factor X (FXa), the FXa hydrolyzes a chromogenic substrate to generate yellow p-nitroaniline (rho NA), the p-nitroaniline has maximum absorption at the wavelength of 405nm, under the condition that the FX and the chromogenic substrate are sufficient, the concentration of the NA rho is related to the activity of the FXA and has linear correlation within a certain range, and the activity of the FX is calculated according to the amount of substances generating the rho NA in unit time. The titer determination method can accurately determine the titer of the FXA, does not need the indirect calculation of the titer of a to-be-detected product by using the FXA and FXa standard substances, reduces errors in the test process, and is accurate, convenient and quick. The method has good repeatability, precision, durability and linearity, and can stably determine the potency of the FXA.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for determining the potency of a factor x activator, the method comprising:
1) diluting the blood coagulation factor X by using a buffer solution A to obtain a solution 1 with the concentration of the blood coagulation factor X of 1.0U/ml-2.0U/ml;
2) diluting a coagulation factor X activator by using a buffer solution B to prepare a solution 2;
3) dissolving a chromogenic substrate with deionized water to obtain a solution 3 with the concentration of 1.5 mg/ml-3.0 mg/ml;
4) uniformly mixing the solution 1, the solution 2 and the buffer solution C, placing the mixture in a water bath at 37 ℃ for heat preservation for 8-12 minutes, adding the buffer solution D and the solution 3, uniformly mixing, measuring the light absorption values of 0 th, 1 th, 2 th and 3 th minutes at the wavelength of 405nm, replacing the solution 2 with the buffer solution B, and operating the same method to serve as a blank control; the volume ratio of the solution 1 to the solution 2 to the solution 3 to the buffer solution C to the buffer solution D is 1: 1;
5) calculating the concentration of p-nitroaniline according to a calculation formula to obtain the potency of the blood coagulation factor X activator;
wherein the buffer solution A is Na+Disodium hydrogen phosphate buffer-sodium dihydrogen phosphate buffer solution with ion content of 0.02 mol/L, tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution with tris (hydroxymethyl) aminomethane content of 0.02 mol/L, or Na+Sodium citrate-citric acid buffer solutions with an ion content of 0.02 mol/L, both containing 0.1 mol/L mol of sodium chloride and 0.001 mol/L mol of benzamidine hydrochloride, and having a pH of 7.4;
the buffer solution B is Na+Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with ion content of 0.01 mol/L, tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution with concentration of 0.01 mol/L based on tris (hydroxymethyl) aminomethane content, or Na+The concentration of the ion content meter is 0.01 mol/L sodium citrate-citric acid buffer solution, and the pH value is 8.0;
the buffer solution C is Na+Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with ion content of 0.1 mol/L, tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution with tris (hydroxymethyl) aminomethane content of 0.1 mol/L, or Na+The sodium citrate-citric acid buffer solution with the ion content of 0.1 mol/L contains 0.3 mol/L sodium chloride, 0.002 mol/L benzamidine hydrochloride and 0.01 mol/L calcium chloride, and the pH value is 7.5;
the buffer solution D is Na+Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with ion content of 1 mol/L, and tris (hydroxymethyl) aminomethaneTris-hydrochloric acid buffer solution with 1 mol/L concentration in terms of alkane content or Na+The sodium citrate-citric acid buffer solution with the ion content of 1 mol/L contains 0.75 mol/L mol of sodium chloride and 0.05 mol/L mol of EDTA-Na2The pH is 8.3;
the concentration of the solution 2 is controlled so that the concentration of the produced rho NA is 0.05 mu mol/ml-1·min-1～0.25μmol·ml-1·min-1；
The calculation formula is
△ASample (A)Representing the difference between the absorbance at the t minute and the absorbance at the 0 minute of the sample, △ AAir conditionerRepresents the difference in absorbance at t minute and 0 minute for the blank; t represents the time of determination of sample and blank control in min, t > 0, preferably t =1, 2 or 3; vtRepresents the total volume of the reaction in ml; 10.4 in ml. mu. mol-1·cm-1The molar absorptivity of rho NA; 1 denotes the optical path length in cm; vsThe test volume of fxa in ml is used.
In one embodiment according to the invention, the concentration of the solution 1 is between 1.0U/ml and 1.5U/ml; preferably 1.25U/ml.
In one embodiment according to the invention, the concentration of said solution 3 is comprised between 1.5mg/ml and 3.0 mg/ml; preferably 1.5 mg/ml.
In one embodiment according to the invention, said factor x is a human or bovine coagulation factor.
In one embodiment according to the invention, the chromogenic substrate is a short peptide containing a p-nitroanilide group (ρ NA) or its hydrochloride.
In one embodiment according to the invention, the factor X activator is derived from snake venom.
In one embodiment according to the invention, the snake venom is selected from one or more of Agkistrodon halys venom, Agkistrodon Halys venom and Vipera pernicialis venom.
The inventor finds that under the condition of sufficient amounts of the coagulation factor X and the chromogenic substrate, the production amount of the p-nitroaniline is only related to the amount of the coagulation factor X activator, and has better linear correlation, and the correlation coefficient R is more than or equal to 0.99. The method has good repeatability, and the RSD is 2.1%. The intermediate precision and the durability are better, and the method comprises that the RSD of different testers is 2.8%, the RSD of chromogenic substrates of different manufacturers is 2.2%, the RSD of blood coagulation factor X of different manufacturers is 6.8%, and the RSD of ultraviolet spectrophotometer of different manufacturers is 2.6%. The recovery of this process was 100.2%. Therefore, the method can accurately and stably test the potency of the blood coagulation factor X activator, is obviously superior to a plasma substrate method and an external standard method of F Xa and F Xa standard products, can provide accurate clinical dosage, and improves the safety and effectiveness of clinical medication.
Drawings
FIG. 1 is a linear plot of the reciprocal of the dilution factor of FXA versus the concentration of rho NA in the determination of the concentration of FXA test.
FIG. 2 is a linear plot of calculated recovery.
Detailed Description
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1Preferred concentrations of coagulation Factor X (FX) and chromogenic substrate
F X selects human source, chromogenic substrate selects S-2765, F X is respectively prepared into solutions of 0.75U/ml, 1.0U/ml, 1.25U/ml, 1.5U/ml and 2.0U/ml by using Tris-HCl buffer solution (containing 0.1 mol/L NaCl and 0.01 mol/L benzamidine hydrochloride), F XA sample is taken as a solution 1, F XA sample is moderately diluted by using Tris-HCl buffer solution (containing pH 8.00.01mol/L) to be taken as a solution 2, chromogenic substrate S-2765 is taken, deionized water is used for dissolving, and the solution is respectively diluted into solution with the concentration of 1.0mg/ml, 1.5mg/ml, 2.0mg/ml, 2.5 mg/ml and 3.0mg/ml, Tris solution 3 is prepared, pH 7.50.1mol/L-HCl buffer solution (containing 0.3 mol/L,002, 0. L mol/ml of benzamidine hydrochloride) to be taken as a solution 3,CaCl20.01 mol/L) as buffer solution C, preparing Tris-HCl buffer solution (containing NaCl 0.75 mol/L and EDTA-Na) with pH of 8.31mol/L20.05 mol/L) as a buffer solution D, pH8.00.01mol/L Tris-HCl buffer solution replaces a FX test article to be used as a blank control, an ultraviolet spectrophotometer is started, 0.2ml of each solution 1, 2 and buffer solution C are respectively added into a cuvette and mixed uniformly when the temperature reaches 37 ℃, the online heat preservation is carried out for 10min at the temperature of 37 +/-0.5 ℃ of the ultraviolet spectrophotometer, 0.2ml of buffer solution D and 0.2ml of solution 3 are added, the mixture is mixed uniformly and continuously kept warm, the absorption values of 0, 1, 2 and 3min at the wavelength of 405nm are detected, the data are substituted into a formula, the concentration of rho NA is calculated, and the preferred concentration results of FX and S-2765 are shown in the following table 1.
Table 1 preferred concentration results for F X and S-2765.
The results show that in the present invention, the concentration of FX is in the range of 1.0U/ml to 2.0U/ml, the concentration of S-2765 is in the range of 1.5mg/ml to 3.0mg/ml, the measurement of the concentration of. rho.NA is stable, and FX and S-2765 are sufficient.
Example 2Determination of the assay concentration of XXA
Preparing solution 1 of 1.25U/ml from FX (human source) with buffer solution pH7.40.02mol/L Tris-HCl (containing 0.1 mol/L NaCl and 0.01 mol/L benzamidine hydrochloride), diluting F XA sample with buffer solution pH8.00.01mol/L Tris-HCl for 20000 times, 12500 times, 8333 times, 5000 times, 4000 times and 3030 times to obtain solution 2, dissolving chromogenic substrate S-2765 in deionized water to obtain solution 1.5mg/ml, dissolving in deionized water to obtain solution 3, and preparing buffer solution pH7.50.2mol/L Tris-HCl (containing NaCl 0.6 mol/L, benzamidine hydrochloride 0.004 mol/L, CaCl 320.02 mol/L) as buffer solution C, preparing Tris-HCl buffer solution (containing NaCl 0.75 mol/L and EDTA-Na) with pH of 8.31mol/L20.05 mol/L) as buffer solution D, pH8.00.01mol/L Tris-HCl buffer solution as blank control instead of FX A test sample, turning on ultraviolet spectrophotometer, adding 0.2ml of each of solution 1, 2 and buffer solution C into colorimeter when temperature reaches 37 deg.CAnd (3) carrying out dish mixing, carrying out online heat preservation for 10min at 37 +/-0.5 ℃ of an ultraviolet spectrophotometer, adding 0.2ml of buffer solution D and 0.2ml of solution 3, mixing uniformly, carrying out continuous heat preservation, detecting the absorption values of 0, 1, 2 and 3 minutes at 405nm wavelength, substituting the data into a formula, and calculating the concentration of rho NA, XXA titer (U/ml) = concentration × dilution times of rho NA.
The concentration range determination results for paranitroaniline (. rho.NA) are shown in Table 2, which in turn defines the optimum working concentration range for FXA.
Table 2F xa concentration determination results.
Drawing a curve (see figure 1) by taking the reciprocal of the dilution factor of the test sample as the abscissa and the rho NA concentration as the ordinate, wherein the linear equation is Y =856.61X +0.00162, R2= 0.99118. As can be seen from the data in Table 2, in the present invention, FXA was diluted to an appropriate concentration so that the concentration of the resulting rho NA was 0.042 to 0.274. mu. mol/ml-1·min-1Within the scope, the potency of fxa is stable. In the present invention, the FXA sample is suitably diluted so that the concentration of the resulting. rho.NA is 0.05. mu. mol/ml-1·min-1～0.25μmol·ml-1·min-1Within the range of (1).
Example 33 batches of F XA potency assay
FX (human source) is prepared into 1.25U/ml solution by using pH7.40.02mol/L Tris-HCl buffer solution (containing 0.1 mol/L NaCl and 0.01 mol/L benzamidine hydrochloride) as solution 1, 3 batches of F XA samples are moderately diluted by using pH8.00.01mol/L Tris-HCl buffer solution to enable the concentration of rho NA to be in the range of 0.05 mu mol/ml to 0.25 mu mol/ml to be used as solution 2, chromogenic substrate S-2765 is prepared, dissolved and diluted into 1.5mg/ml solution by using deionized water to be used as solution 3, and pH7.50.2mol/L Tris-HCl buffer solution (containing 0.6 mol/L NaCl, 0.004 mol/L benzamidine hydrochloride, CaCl20.02 mol/L) as buffer solution C, preparing Tris-HCl buffer solution (containing NaCl 0.75 mol/L and EDTA-Na) with pH of 8.31mol/L20.05 mol/L) as buffer solution D, pH8.00.01mol/L Tris-HCl buffer instead of FXA test sample was used as a blank control. The UV spectrophotometer was turned on until the temperature reached 37 ℃. Respectively taking 0.2ml of each of the solutions 1, 2 and the buffer solution C, adding the solutions into a cuvette, mixing the solutions uniformly, carrying out online heat preservation for 10min at the temperature of 37 +/-0.5 ℃ of an ultraviolet spectrophotometer, adding 0.2ml of the buffer solution D and 0.2ml of the solution 3, mixing the solutions uniformly, continuing heat preservation, and detecting the absorption values of 0, 1, 2 and 3 minutes at the wavelength of 405 nm. The data were substituted into the formula to calculate the concentration of ρ NA. The formula is as follows:
titers were calculated as batch No. 150107F XA, VtRepresenting a total volume of 1ml, VsDenotes the volume of the XXA sample of 0.2ml, t denotes the measurement time of the sample and the blank of 3min, △ ASample (A)The difference between the absorbance at the 3 rd minute and the absorbance at the 0 th minute of the sample is 1.092, △ AAir conditionerThe difference between the absorbance values at the 3 rd minute and the 0 th minute of the blank was 0.000, and the concentration of rho NA was calculated by substituting the difference into the formula to be 0.175. mu. mol. ml-1·min-1And the dilution multiple of the F XA of the 150107 batches is 5000, so that the titer is 875U/ml, the protein content is 0.155mg/ml, and the specific activity is 875/0.155= 5645U/mg.
In the present invention, the F XA activity unit is defined as: the amount of enzyme required to form 1. mu. mol of p-nitroaniline at 37 ℃ at pH8.0 in 1min was defined as 1 unit.
The factor X activator titers were determined in 3 batches as described in example 3, and the results are shown in Table 3.
Table 33 results of potency assay of batch F xa.
Example 4Repeatability of the XXA potency assay
The fxa titer was determined as in example 3, taking test sample 150107 for testing, repeating 6 times, and recording the data, with the results as in table 4.
Table 4F xa potency assay reproducibility results.
Example 5FXA potency assay methods intermediate precision investigation
The XXA titer was determined as in example 3, taking sample 150107, testing at different times by different persons and recording the test data, the results are shown in Table 5.
Table 5 fxa potency assay methods intermediate precision results.
Example 6Fxa potency assay durability test
1) Determination of F XA titer stability by chromogenic substrate S-2765 of different manufacturers
The test sample 150107 was tested as in example 3, the titer was determined using chromogenic substrate S-2765 from a different manufacturer, and the results are reported in Table 6.
Table 6 results of F XA titer determination by different manufacturers S-2765.
2) Stability of assay of F X A potency of reaction substrate F X of different manufacturers
The FXA titer was determined as in example 3, taking the test sample 150107, measuring with the reaction substrate FX of different manufacturers, and recording the test data, the results are shown in Table 7.
Table 7 results of F xa titer determination by different manufacturers F x.
3) Stability for measuring XXA titer by different types of ultraviolet spectrophotometers
The XXA titer was measured as in example 3, the test sample 150107 was used, the titer was measured by UV spectrophotometer of different types, and the results are shown in Table 8.
Table 8 results of F xa titer measurements with different instrument types.
Example 7Linearity and accuracy (recovery) of the assay for F XA potency
The F XA titer was measured by the method of example 3, and the test sample 150107 was diluted 8333 times, 7143 times, 6250 times, 5000 times and 4000 times, respectively, and the test data was recorded, and the results are shown in Table 9.
Table 9 linearity and recovery results.
Drawing a curve (see fig. 2) by taking the reciprocal of the dilution factor of the test sample as the abscissa and the rho NA concentration as the ordinate, wherein the linear equation is y =848.13x + 0.00282R2=0.99356, linearity is good.
And substituting the reciprocal of the dilution factor into a standard curve, calculating theoretical titer, wherein the ratio of the actual titer to the theoretical titer is the recovery rate, the average recovery rate is 100.2%, RSD =2.6%, and the method has good accuracy.
Example 8Comparison of the assay results of the XXA titer assay of the present invention with that of the direct plasma-based assay
1) Potency determination of FXA by using common human plasma as direct substrate method
A test sample F XA, batch No. 150107, is diluted 800 times by 0.01 mol/L Tris-HCl buffer solution with pH8.0 to be used as a test sample solution, ordinary human plasma (blood station source) is taken, water bath at 37 ℃ is completely melted for later use, and deionized water is used for preparing a calcium chloride solution with the concentration of 0.02 mol/L for later use.
0.2ml of ordinary human plasma is taken into a small test tube and preheated for 2min in a 37-test way, 0.1ml of test solution is added, 0.1ml of 0.02 mol/L calcium chloride solution is added, the mixture is shaken up and timed, the time of white flocs of the plasma is recorded, and the measurement results are shown in table 10.
As can be seen from Table 10 below, the results of the test were very different between the plasma and the FXA titer could not be accurately and stably tested.
Table 10 results of F xa titer determination using common human plasma as a direct substrate.
2) Potency of FXA determined by using human coagulation quality control plasma as direct substrate method
A test sample F XA, batch No. 150107, is diluted 800 times by 0.01 mol/L Tris-HCl buffer solution with pH8.0 to be used as a test sample solution, human blood coagulation quality control plasma (purchased from two manufacturers respectively) is taken from each manufacturer, 2 batches are respectively dissolved by 1ml of deionized water for later use, and deionized water is used for preparing a calcium chloride solution with the concentration of 0.02 mol/L for later use.
0.2ml of human coagulation quality control plasma is taken to be put into a small test tube and preheated for 2min at 37, 0.1ml of test solution is added, 0.1ml of 0.02 mol/L calcium chloride solution is added, the mixture is shaken up and timed, the time of white flocs of the plasma is recorded, and the measurement results are shown in Table 11.
As can be seen from table 11 below, the test results of different batches of plasma from the same manufacturer and the test results of different manufacturers are different, and the fxa titer cannot be accurately and stably tested.
Table 11 results of F xa titer determination using human coagulation plasma as a direct substrate.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.
Claims (9)
1. A method for determining the potency of a factor X activator, comprising: the method comprises the following steps:
1) diluting the blood coagulation factor X by using a buffer solution A to obtain a solution 1 with the concentration of the blood coagulation factor X of 1.0U/ml-2.0U/ml;
2) diluting a coagulation factor X activator by using a buffer solution B to prepare a solution 2;
3) dissolving a chromogenic substrate with deionized water to obtain a solution 3 with the concentration of 1.5 mg/ml-3.0 mg/ml; the chromogenic substrate is short peptide containing p-nitroaniline group or hydrochloride thereof;
4) uniformly mixing the solution 1, the solution 2 and the buffer solution C, placing the mixture in a water bath at 37 ℃ for heat preservation for 8-12 minutes, adding the buffer solution D and the solution 3, uniformly mixing, measuring the light absorption values of 0, 1, 2 and 3 minutes by using an ultraviolet spectrophotometer at the wavelength of 405nm, replacing the solution 2 with the buffer solution B, and operating by the same method to serve as a blank control; the volume ratio of the solution 1 to the solution 2 to the solution 3 to the buffer solution C to the buffer solution D is 1: 1;
5) calculating the concentration of p-nitroaniline according to a calculation formula to obtain the potency of the blood coagulation factor X activator;
wherein the buffer solution A is Na+Disodium hydrogen phosphate buffer-sodium dihydrogen phosphate buffer solution with ion content of 0.02 mol/L, tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution with tris (hydroxymethyl) aminomethane content of 0.02 mol/L, or Na+Sodium citrate-citric acid buffer solutions with an ion content of 0.02 mol/L, both containing 0.1 mol/L mol of sodium chloride and 0.001 mol/L mol of benzamidine hydrochloride, and having a pH of 7.4;
the buffer solution B is Na+Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with ion content of 0.01 mol/L, tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution with concentration of 0.01 mol/L based on tris (hydroxymethyl) aminomethane content, or Na+The concentration of the ion content meter is 0.01 mol/L sodium citrate-citric acid buffer solution, and the pH value is 8.0;
the buffer solution C is Na+Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with ion content of 0.1 mol/L, and tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution with tris (hydroxymethyl) aminomethane content of 0.1 mol/LWith Na+The sodium citrate-citric acid buffer solution with the ion content of 0.1 mol/L contains 0.3 mol/L sodium chloride, 0.002 mol/L benzamidine hydrochloride and 0.01 mol/L calcium chloride, and the pH value is 7.5;
the buffer solution D is Na+Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with ion content of 1 mol/L, tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution with tris (hydroxymethyl) aminomethane content of 1 mol/L, or Na+The sodium citrate-citric acid buffer solution with the ion content of 1 mol/L contains 0.75 mol/L mol of sodium chloride and 0.05 mol/L mol of EDTA-Na2The pH is 8.3;
the concentration of the solution 2 is controlled so that the concentration of the produced rho NA is 0.05 mu mol/ml-1·min-1～0.25μmol·ml-1·min-1；
The calculation formula is
△ASample (A)Representing the difference between the absorption values of the samples at the t minute and at the 0 minute △ AAir conditionerRepresents the difference between the absorbance of the blank at minute t and minute 0; t represents the time for the determination of the sample and blank control in min, t > 0; vtRepresents the total volume of the reaction in ml; 10.4 in ml. mu. mol-1·cm-1The molar absorptivity of rho NA; 1 denotes the optical path length in cm; vsThe test volume of fxa in ml is used.
2. The method of claim 1, wherein the concentration of solution 1 is 1.0U/ml to 1.5U/ml.
3. The method of claim 1 or 2, wherein the concentration of solution 1 is 1.25U/ml.
4. The method of claim 1, wherein the concentration of solution 3 is 1.5mg/ml to 3.0 mg/ml.
5. The method of claim 4, wherein the concentration of solution 3 is 1.5 mg/ml.
6. The method of claim 1, wherein t =1, 2, or 3 in the formula.
7. The method of claim 1, wherein said factor X is of human or bovine origin.
8. A method according to claim 1, wherein the factor x activator is derived from snake venom.
9. The method of claim 8, wherein the snake venom is selected from one or more of agkistrodon spearhead venom, agkistrodon pustulosa venom, and viper venom.
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| 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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| 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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| 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;李晶等;《中国新药杂志》;20070923;第16卷(第18期);第1491-1495页 * |
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