CN102692511A - Kit (Developing substrate method) for testing protein C (PC) - Google Patents
Kit (Developing substrate method) for testing protein C (PC) Download PDFInfo
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Abstract
The invention discloses a kit for detecting the activity of a protein C. The kit for detecting the activity of the protein C provided by the invention comprises a protein C activating agent obtained by separating and purifying venom of vipers in Qinling Mountains of China, and a developing substrate reagent, and belongs to detection by a developing substrate method. The kit has good detection specificity and is not easy to be interfered; and raw materials are easy to obtain, the preparation is simple, the operation is simple and fast in a detection process, and an instrument applicable range is wide, so that the kit is convenient to popularize and use in each level of hospitals, hygiene departments and the like.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of chromophoric substrate method detects the active kit of PROTEIN C.
Background technology
PROTEIN C (being called for short PC) is a kind of vitamin k-dependent protein, at Ca
2+Under the condition that exists, can be regulated the albumen activation by fibrin ferment-fibrin ferment and change into activated PROTEIN C (being called for short APC).APC can be through proteolysis deactivation activation prothrombinase (being called for short the F V a) (is called for short the F VIII a), thereby demonstrates stronger anticoagulating active with VIII a; Promote fibrinolytic through discharging the blood vessel plasminogen activators simultaneously.The shortage of PC can make the blood coagulation of human body and fibrinolytic balance influenced, makes blood coagulation hyperfunction, thereby thrombotic diseases takes place.
The reduction of blood plasma PC level is prone to cause disseminated intravascular coagulation (DIC) and liver diseases, like cirrhosis and chronic hepatitis.In recent years, it is found that many clinical diseases all can follow the change of PC level, like renal failure, operation, inflammatory reaction, oral antivitamin K class anti-freezing medicine, ischemic heart disease, cranial vascular disease, venous thrombosis and leukaemia etc.This shows that necessary foundation a kind ofly can accurately be detected the active short-cut method of PC in the blood plasma, is used for the auxiliary diagnosis of above-mentioned all kinds of diseases.
According to detecting principle, the method that is used for the detection by quantitative PROTEIN C at present can reduce three major types:
(1) immunological method that forms according to the antigenic characteristic of PC; Comprise double-antibody sandwich enzyme linked immunosorbent assay (ELISA), radio immunoassay, rocket immunoelectrophoresis etc.; Wherein the most widely used is the ELISA method, through adopting monoclonal antibody or manyly anti-ly carrying out antigen-antibody reaction and detect the PC antigenic content.This method can accurately detect the PC antigenic content, but exists certain defective.At first, need highly purified PC to obtain specific antibody, because contained plasma proteins impurity can cause the selectivity of antibody not strong in the impure antigen, this can make that testing result is higher; Secondly, this method complex operation, detection time is very long, can not satisfy the needs of diagnosis in time of emergency treatment and clinical patient and treatment; At last, this method also has certain limitation, and it has also detected the pathomorphism of PC simultaneously, like PC that be connected with suppressant or deactivation, therefore can not judge accurately whether the PC that is detected has normal activity.
(2) the APTT freezing method that forms according to the PC anticoagulating active.This method joins in the clotting of plasma system after using snake venom to activate PC, can prolong PCT, the prolongation ratio corresponding PC content in the sample.This method ability selectivity detection functionality PC, but the disturbing factor of this method is many, the testing result parameter is bigger, and its result's judgement is often decided with reviewer's experience, and directly result's accuracy and repeatability is measured in influence.
(3) adopt the Enzymology method of the narrow spectrum synthetic polypeptide of APC as substrate, i.e. chromophoric substrate method.Early stage fibrin ferment-the fibrin ferment that adopts is regulated albumen (T-TM) as activator.The PC that this method can not directly detect in the blood plasma is active, because include PC suppressant and other interfering materials in the blood plasma, is prone in active testing process, react with synthetic peptide substrates.Therefore, must through antibody column or absorption PC be separated from blood plasma in advance, this process not only needs a large amount of blood plasma, and needs great amount of time and manpower, and this method is not suitable for the fast processing of batch samples.At present, abroad developed and used the snake venom activator to replace T-TM, it can quick active people and Ox blood plasma PC, activation mechanism maybe with thrombin class seemingly, but do not receive the interference of other clotting factor, hydrolysis substrate not yet.Therefore it is minimum that its quick active effect makes the effect of PC suppressant reduce to, and got rid of the necessity of adsorptive separation PC, thereby quicken PC reactivation process, and it is oversize to have overcome when making activator with T-TM activationary time, the defective of complex operation.
At present, the method that the PROTEIN C active level detects mainly adopts the chromophoric substrate method, and this method can reflect accurately, specifically that PC is active, and not only real result is stable, and easy to operation, has advantage fast, only needs several minutes just can accomplish detection.But owing to the activator overwhelming majority who is adopted in this type of at present commercially available kit is that separation and purification gets from Aneistrodon piscivorus (Agkistrodon contortrix) poison; This kind snake is distributed in southeastern US and Mexico northeast more; Domestic acquisition for this snake venom is relatively more difficult, and therefore this activator can't be produced in enormous quantities and use at home.In the recent period; There is bibliographical information from the viper venom of south, Anhui, to separate the PC activator of purifying and has the prospect that is used for the active detection of clinical PC; But this activator of experiment proof has part to activate to other clotting factor in the blood plasma; Be subject to multiple factor and disturb, specificity is relatively low, and this activator also can not be applied in the PC mensuration kit at present.
More than all drawbacks make PC detect to be difficult to promote the use of in hospital, thereby the routine that has limited this test item is carried out.Therefore, press for a kind of new PC activator of exploitation and be used for PC determination of activity kit.
Summary of the invention
The object of the present invention is to provide the active detection kit of a kind of PROTEIN C, this kit is based on Chinese Qinling Mountains viper venom activator, and effectively detection by quantitative plasma proteins C is active, and its detection specificity is strong; And raw material is easy to get, cost is lower, is fit to mass production.
Particularly, the invention provides a kind of active kit of PROTEIN C that detects, this kit comprises R1 reagent and R2 reagent; Said R1 reagent comprises a kind of PROTEIN C activator, and said PROTEIN C activator extracts in the viper venom of the Chinese Qinling Mountains, and its SDS-PAGE electrophoresis is a band, and molecular weight is between 40-45KD; Said R2 reagent is the reagent that comprises chromophoric substrate.
In the mentioned reagent box, the method that said PROTEIN C activator extracts in the viper venom of the Chinese Qinling Mountains is following:
Step 1: after Chinese Qinling Mountains viper venom dry powder dissolves with phosphate buffer; Adopt the fast flow velocity anion-exchange column of DEAE-agarose to carry out chromatography; Phosphate buffer with containing 0～0.5mol/L sodium chloride carries out linear gradient elution; The detection wavelength is 280nm, collects and merges active peak eluent;
Step 2: with the active peak of step 1 gained eluent behind the dialysis desalination; Adopt SP-Sephadex C50 cation exchange column to carry out chromatography; Acetate buffer with containing 0.1～0.6mol/L sodium chloride carries out linear gradient elution, and the detection wavelength is 280nm, collects and merges active peak eluent;
Step 3: the active peak of step 2 gained eluent adopts Sephadex G-100 post to carry out filtration chromatography behind the desalination that concentrates, dialyses, and the phosphate buffer wash-out is collected active peak eluent, is said PROTEIN C activator.
In the said extracted method, the pH value of the said phosphate buffer of step 1 is 7～8, concentration is 10～25mM.
The said Chinese Qinling Mountains of step 1 viper venom dry powder is counted 0.5～1.5:10 with the mass volume ratio of the phosphate buffer that is used to dissolve snake venom dry powder with g/ml.
The pH value of the said acetate buffer of step 2 is 5～6, concentration is 30～60mM.
The pH value of the said phosphate buffer of step 3 is 7～8, concentration is 10～30mM.
In the mentioned reagent box, said R1 reagent and R2 reagent also comprise damping fluid, stabilizing agent, excipient and antiseptic respectively.
In the mentioned reagent box, the working concentration of PROTEIN C activator is 0.07～0.36mg/ml described in the said R1 reagent.
Chromophoric substrate is PyroGlu-Pro-Arg-pNAHCl in the said R2 reagent, and working concentration is 0.1～0.5mg/ml.
Said R1 reagent damping fluid is Tris damping fluid or HEPES damping fluid, for reaction provides suitable reaction conditions.
Preferably, said R1 reagent damping fluid is that the pH value is 7.5～8.5, concentration is the Tris-HCl damping fluid of 4～6mmol/L.
Said R2 reagent damping fluid is one or more in Tris-HCl damping fluid, Tris-CsCl damping fluid, Tris-NaCl damping fluid, imidazole buffer, HEPES damping fluid, the barbitol buffer solution.
Preferably, said R2 reagent damping fluid is that the pH value is 6.9～8.5 Tris-CsCl damping fluid, and said Tris final concentration is 50～100mmol/L, and said Cs+ final concentration is 100～260mmol/L.
As preferably, also contain Ca in the said R2 reagent damping fluid
2+, can quicken substrate reactions speed, said Ca
2+Final concentration is 4～6mmol/L.
In the kit of the present invention, the stabilizing agent in said R1 reagent, the R2 reagent is one or more in protein, amino acid, surfactant, suspending agent, the antioxidant; Said protein is bovine serum albumin(BSA) or gelatin, and said surfactant is PEG-6000 or PEG-8000.
Excipient in said R1 reagent, the R2 reagent all is selected from one or both in sucrose, sweet mellow wine, glycocoll, trehalose, the lactose.
Antiseptic in said R1 reagent, the R2 reagent is the conventional preservatives of this area, and like Sodium azide, when content was 0.02%-0.2% (g/ml), both effectively sterilizations can not have side effects again, can prolong the term of validity of kit; Can also be any in other gentamicin, Ciprofloxacin or the ProClin series antiseptic with antisepsis.
Kit of the present invention has following advantage:
1, the PROTEIN C activator in the kit of the present invention derives from Chinese Qinling Mountains viper venom, and domestic being easy to of starting material obtains, and the preparation method is easy, is easy to industrialization production;
2, the PROTEIN C activator in the kit of the present invention does not have activation to other clotting factor in the blood plasma, has stronger selectivity, and therefore is not subject to disturb, and has stronger specificity;
3, kit reagent of the present invention is formed simply, is easy to preparation, and production cost is lower;
4, to detect PROTEIN C simple to operate when active for kit of the present invention, is applicable to the semi-automatic or full automatic blood coagulation analyzer of multiple model, is convenient to promote the use of in hospitals at different levels, medical biotechnology R&D institution etc., thereby can promotes the routine of this test item to carry out.
Description of drawings
Fig. 1 is the calibration curve of kit of the present invention;
Fig. 2 is the correlation analysis of kit of the present invention and existing PROTEIN C detection kit.
Embodiment
The invention discloses the active detection kit of a kind of PROTEIN C, those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is done further detailed description below in conjunction with specific embodiment.Concrete material proportion, process conditions and result thereof described in the embodiment only are used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
The preparation of embodiment 1 PROTEIN C activator
(1) gets 1g Qinling Mountains pallas pit viper raw venin dry powder (available from the Ning Dong of the Shaanxi Province She Chang of forestry bureau); Be dissolved in 10ml 20mM PBS damping fluid (pH7.5); Leave standstill, get supernatant and join in the DEAE-Sepharose FF anion-exchange column of using the same buffer balance in advance, after last appearance is accomplished; Wash pillar once more to balance with above-mentioned damping fluid, so that remove all the other foreign proteins that are not attached on the pillar.Carry out linear gradient elution with the same buffer that contains 0～0.5mol/L NaCl again, flow velocity is 1ml/min, and the detection wavelength is 280nm.Get the 0.1ml eluent, add the normal pooled plasma of 0.1ml and 37 ℃ of warm in advance 0.1ml APTT reagent (cephalin-white bole), hatch 5min for 37 ℃ behind the mixing, add 37 ℃ of 0.025mol/L CaCl of temperature in advance then
2Behind the solution 0.1ml, measure setting time, as prolong, explain that eluent contains the active component of ability activated protein c, collect and merge active peak eluent.
(2) the active peak eluent of step (1) being collected is with pH 5.8,50mM NaAc-HAc damping fluid dialysis desalination, and last kind on the SPSephadex C-50 cation exchange column after the same buffer balance, and flow velocity is 1ml/min.Last appearance is washed pillar to balance with above-mentioned damping fluid after accomplishing once more, and the employing NaAc-HAc damping fluid that contains 0.1～0.6mol/L NaCl carries out linear gradient elution then, collects and merges active peak eluent.
(3) after the active peak eluent of step (2) being collected concentrates with polyglycol-20000; Move in the bag filter and dialyse once more, dislysate is the PBS damping fluid of concentration 20mM, pH8.0, fully after the dialysis; Last appearance is on the Sephadex G-100 chromatographic column after the abundant balance of same buffer; Flow velocity is 0.4ml/min, carries out wash-out with this level pad with identical flow velocity, collects the about 10ml in active peak.At last, collected activated protein dialysis back is concentrated, the packing freeze-drying is preserved.With reductibility and irreducibility SDS-PAGE electrophoresis detection, all be shown as a band, molecular weight is between 40 ~ 45kD.
The preparation of embodiment 2 kits of the present invention
PROTEIN C determination of activity kit in the present embodiment is a solid reagent, comprises R1 reagent and R2 reagent, respectively by following composition and consumption preparation:
A) R1 reagent
Mentioned reagent is all after the dissolving fully, with the 1ml/ bottle branch bottle of packing into, uses after processing freeze-dried powder.
B) R2 reagent
Mentioned reagent is all after the dissolving fully, with the 1ml/ bottle branch bottle of packing into, uses after processing freeze-dried powder.
Embodiment 3 kits of the present invention detect the active method of PROTEIN C
(available from U.S. ANIARA company, article No.: A222101), be mixed with the calibration object solution of 6 different activities with physiological saline, activity is respectively 150%, 100%, 75%, 50%, 25%, 0% to get the PROTEIN C calibration object.Solid R1 reagent in the embodiment 2 prepared kits is used the 1ml dissolved in distilled water, and R2 reagent is used the 1ml dissolved in distilled water.Be operating as example with the full-automatic coagulo meter of Japanese East Asia CA530: set 37 ℃ of temperature of reaction, the mensuration wavelength is 405nm, gets the calibration object solution 20 μ l of variable concentrations respectively; Behind the preheating 60s, add R1 reagent 125 μ l, add R2 reagent 30 μ l behind the reaction 300s; Absorbance difference when assaying reaction 11s and 100s (△ OD); Every pipe replication 3 times, the average of the absorbance △ OD that each calibration tube is recorded for 3 times is an ordinate, corresponding activity is a horizontal ordinate; Make " activity-absorbance difference " calibration curve, the result sees Fig. 1.
Get testing sample, the absorbance △ OD value of the same method working sample, the substitution calibration curve can calculate the activity of PROTEIN C in the testing sample.If the detection of active of PROTEIN C exceeds the scope of calibration curve in the sample, for the accuracy that guarantees to detect, row detection again after need suitably diluting with physiological saline.
This kit is not only applicable to Japanese East Asia CA530 coagulo meter; But also be applicable to the blood coagulation analyzer of other brand, model; Like U.S. Beckman Ku Erte ACL 7000 full-automatic coagulo meters, the semi-automatic coagulo meter of U.S. Pacific Ocean ThromboScreen 400C, the full-automatic coagulo meter of Beijing Sai Kexide SF-8000 etc., concrete parameter can suitably be adjusted according to the instrument difference.
The specificity analyses of embodiment 4 kits of the present invention
In order to investigate the specificity of kit of the present invention, present embodiment has adopted following three kinds of different experiments to come illustrated together to the used snake venom PC of kit of the present invention activator.
1, echidnotoxin C activator (being called for short PCA) is to the influence of human plasma PT and APTT
The snake venom activator some can influence blood coagulation system endogenous approach or (with) exogenous route, and desirable PROTEIN C activator should be able to suppress the endogenous approach of blood coagulation system, to not influence of exogenous route.This experiment is analyzed the influence of people's normal plasma APTT and PT through research the present invention used Qinling Mountains viper venom PCA.
Above-mentioned snake venom PCA with variable concentrations carries out PT and APTT investigation, and assay method is following:
(1) get the normal pooled plasma of 0.1ml, add 0.1ml snake venom PCA, hatch 5min for 37 ℃, add 37 ℃ of preparatory temperature PT reagent 0.2ml then, the record setting time is the PT value.
(2) get the normal pooled plasma of 0.1ml, add 37 ℃ of 0.1ml APTT reagent (cephalin-white bole) of temperature in advance, and behind the 0.1ml snake venom PCA mixing, hatch 5min for 37 ℃, add 37 ℃ of 0.025mol/L CaCl of temperature in advance then
2Behind the solution 0.1ml, the record setting time is the APTT value.
Mensuration result is as shown in table 1.
The used snake venom PCA of table 1 the present invention is to the influence of PT, APTT
Table 1 data presentation: the adding of the used snake venom PCA of the present invention can cause the prolongation of APTT at once, and time expand and PCA concentration have certain compliance and increases progressively relation; Snake venom PCA concentration be 0.25mg/ml when following to not influence of PT.
2, in the PC antibody and the experiment
Anti-PROTEIN C antibody preparations (available from the Abcam company) solution of different volumes is joined in the normal plasma, hatch 5min for 37 ℃, to simulate weary PC blood plasma.Be sample with physiological saline, normal plasma and each weary PC blood plasma model respectively,, detect respectively with embodiment 2 prepared kits and the kit that substitutes snake venom PCA of the present invention with south, Anhui cobra-venom PCA according to embodiment 3 said detection methods.The result is as shown in table 2.
Show in the 2PC antibody and experimental result
Table 2 data presentation: when being sample with physiological saline, south, Anhui cobra-venom PCA reagent absorbance is slightly larger than snake venom PCA reagent of the present invention; Increase along with PC AC in the blood plasma; △ OD value obviously descends; When PC antibody reached finite concentration, snake venom PCA reagent of the present invention almost can be eliminated the color development reaction that PCA causes fully, and the absorbance difference of south, Anhui cobra-venom PCA reagent mensuration sample is still bigger.
Conclusion: the PCA in the kit of the present invention can not produce the color development reaction by the direct hydrolysis substrate, and only PC produces the color development effect in the blood plasma through activating, and has stronger selectivity; Other clotting factor causes the color development effect in the blood plasma and south, Anhui cobra-venom PCA also possibly partly activate.
3, in the blood plasma other blood coagulation correlative factor to the influence of kit of the present invention
With various weary factor blood plasma and heparinize blood plasma is sample; According to embodiment 3 said detection methods; PC with the prepared kits of embodiment 2 detect in the samples is active, and with existing commercial reagent box (available from Austrian Technoclone company, article No.: 5341013) compare.Detection of active < 80% the interference that is considered to receive plasma coagulation factors.Testing result is as shown in table 3.
The weary factor blood plasma of table 3 is to the influence of kit of the present invention
The result shows, existing commercial reagent box receives the interference of factor IX and XII in the blood plasma to a certain extent, and snake venom PCA of the present invention is all insensitive to it.Therefore, kit of the present invention is compared with commercially available similar kit, more is not vulnerable to the interference of other factors, has stronger specificity.
The correlation analysis that embodiment 5 kits of the present invention and existing commercial reagent box detect plasma sample at random
Totally 33 parts of random collecting normal person and patient's plasma samples, wherein male 18 examples, women 15 examples.(article No.: OUVV15) respectively to sample replication 2 times, calculate respectively and measure average, calculate both related coefficients, the line linearity of going forward side by side returns to measure kit with kit of the present invention and German Siemens company PROTEIN C; Its testing result is seen correlation coefficient r=0.993 of 2, two kinds of kits of accompanying drawing, and equation of linear regression is y=0.9886x+1.7322.γ＞0.975 that requires according to standardization body of U.S. clinical labororatory (CLSI) file draws: the testing result that kit of the present invention and German Siemens company PROTEIN C are measured kit has good consistance, explain that kit of the present invention has the effectiveness that is equal to commercially available import reagent box in the active detection of PROTEIN C.After kit of the present invention puts goods on the market, can realize import substitution, reduce and detect cost, help applying at various big hospital.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (10)
1. one kind is detected the active kit of PROTEIN C, it is characterized in that, comprises R1 reagent and R2 reagent; Said R1 reagent comprises a kind of PROTEIN C activator, and said PROTEIN C activator extracts in the viper venom of the Chinese Qinling Mountains, and its SDS-PAGE electrophoresis is a band, and molecular weight is between 40-45KD; Said R2 reagent is the reagent that comprises chromophoric substrate.
2. kit as claimed in claim 1 is characterized in that, the method that said PROTEIN C activator extracts in the viper venom of the Chinese Qinling Mountains is following:
Step 1: after Chinese Qinling Mountains viper venom dry powder dissolves with phosphate buffer; Adopt the fast flow velocity anion-exchange column of DEAE-agarose to carry out chromatography; Phosphate buffer with containing 0～0.5mol/L sodium chloride carries out linear gradient elution; The detection wavelength is 280nm, collects and merges active peak eluent;
Step 2: with the active peak of step 1 gained eluent behind the dialysis desalination; Adopt SP-Sephadex C50 cation exchange column to carry out chromatography; Acetate buffer with containing 0.1～0.6mol/L sodium chloride carries out linear gradient elution, and the detection wavelength is 280nm, collects and merges active peak eluent;
Step 3: the active peak of step 2 gained eluent adopts Sephadex G-100 post to carry out filtration chromatography behind the desalination that concentrates, dialyses, and the phosphate buffer wash-out is collected active peak eluent, is said PROTEIN C activator.
3. kit as claimed in claim 2 is characterized in that, the pH value of the said phosphate buffer of step 1 is 7～8, concentration is 10～25mM.
4. kit as claimed in claim 2 is characterized in that, the said Chinese Qinling Mountains of step 1 viper venom dry powder is counted 0.5～1.5:10 with the mass volume ratio of the phosphate buffer that is used to dissolve snake venom dry powder with g/ml.
5. kit as claimed in claim 2 is characterized in that, the pH value of the said acetate buffer of step 2 is 5～6, concentration is 30～60mM.
6. kit as claimed in claim 2 is characterized in that, the pH value of the said phosphate buffer of step 3 is 7～8, concentration is 10～30mM.
7. like each described kit of claim 1-6, it is characterized in that said R1 reagent and R2 reagent also comprise damping fluid, stabilizing agent, excipient and antiseptic respectively.
8. kit as claimed in claim 7 is characterized in that, the working concentration of PROTEIN C activator is 0.07～0.36mg/ml described in the said R1 reagent.
9. kit as claimed in claim 7 is characterized in that, chromophoric substrate is PyroGlu-Pro-Arg-pNAHCl in the said R2 reagent, and working concentration is 0.1～0.5mg/ml.
10. kit as claimed in claim 7 is characterized in that, said R1 reagent damping fluid is Tris damping fluid or HEPES damping fluid.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103018188A (en) * | 2012-12-28 | 2013-04-03 | 中国医学科学院输血研究所 | Method for detecting human protein C activity |
| CN107167439A (en) * | 2017-06-30 | 2017-09-15 | 上海贞元诊断用品科技有限公司 | A kind of kit that dabigatran content is detected based on Chromogenic assay |
| CN107255622A (en) * | 2017-06-30 | 2017-10-17 | 上海贞元诊断用品科技有限公司 | A kind of kit that dabigatran content is detected based on fibrin ferment Chromogenic assay |
| CN110714051A (en) * | 2019-11-20 | 2020-01-21 | 迈克生物股份有限公司 | Protein C activity determination kit |
| CN113913491A (en) * | 2021-10-10 | 2022-01-11 | 青岛大学附属医院 | Method for measuring protein C activity, reagent for measurement, and method for producing the same |
| CN114814245A (en) * | 2022-06-30 | 2022-07-29 | 深圳市帝迈生物技术有限公司 | Protein C activity determination kit based on chromogenic substrate method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4849403A (en) * | 1985-05-29 | 1989-07-18 | Pentapharm Ag | Protein C activator, methods of preparation and use thereof |
| SU1565889A1 (en) * | 1988-04-18 | 1990-05-23 | Институт Химической И Биологической Физики Ан Эсср | Method of obtaining exogenous activator of protein c |
| WO1993007491A1 (en) * | 1991-10-04 | 1993-04-15 | Board Of Regents Of The University Of Nebraska | A soluble thrombomodulin-based one-stage assay for vitamin k-dependent coagulation-inhibiting proteins |
| EP1068536A1 (en) * | 1998-02-02 | 2001-01-17 | Gradipore Limited | Improved blood coagulation test |
| WO2002079375A1 (en) * | 2001-03-30 | 2002-10-10 | Coagulation Diagnostics, Incorporated | Rapid assessment of coagulation activity in whole blood |
| CN1847262A (en) * | 2005-04-13 | 2006-10-18 | 上海医药工业研究院 | One kind of protein and its use |
-
2012
- 2012-06-08 CN CN201210189574.9A patent/CN102692511B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4849403A (en) * | 1985-05-29 | 1989-07-18 | Pentapharm Ag | Protein C activator, methods of preparation and use thereof |
| SU1565889A1 (en) * | 1988-04-18 | 1990-05-23 | Институт Химической И Биологической Физики Ан Эсср | Method of obtaining exogenous activator of protein c |
| WO1993007491A1 (en) * | 1991-10-04 | 1993-04-15 | Board Of Regents Of The University Of Nebraska | A soluble thrombomodulin-based one-stage assay for vitamin k-dependent coagulation-inhibiting proteins |
| EP1068536A1 (en) * | 1998-02-02 | 2001-01-17 | Gradipore Limited | Improved blood coagulation test |
| WO2002079375A1 (en) * | 2001-03-30 | 2002-10-10 | Coagulation Diagnostics, Incorporated | Rapid assessment of coagulation activity in whole blood |
| CN1847262A (en) * | 2005-04-13 | 2006-10-18 | 上海医药工业研究院 | One kind of protein and its use |
Non-Patent Citations (5)
| Title |
|---|
| 孙林光等: "国产蛇毒激活血浆蛋白C的研究", 《中国病理生理杂志》 * |
| 张世等: "皖南蝮蛇毒PCA活化血浆蛋白C的特性研究", 《皖南医学院学报》 * |
| 张根葆等: "五步蛇毒蛋白C激活剂的纯化与活性分析", 《蛇志》 * |
| 王湘君等: "蝮蛇的形态、生态及命名的研究", 《琼州学院学报》 * |
| 章俊等: "蝮蛇毒蛋白C激活物的纯化与活性分析", 《中国输血杂志》 * |
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