CN103361398A - Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip - Google Patents
Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip Download PDFInfo
- Publication number
- CN103361398A CN103361398A CN201310282186XA CN201310282186A CN103361398A CN 103361398 A CN103361398 A CN 103361398A CN 201310282186X A CN201310282186X A CN 201310282186XA CN 201310282186 A CN201310282186 A CN 201310282186A CN 103361398 A CN103361398 A CN 103361398A
- Authority
- CN
- China
- Prior art keywords
- gly
- val
- arg
- polypeptide
- array chip
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting thrombin activity and screening a thrombin inhibitor in plasma by using a polypeptide microarray chip, which solves the technical problem that the thrombin detection method in the existing analytical chemistry is inapplicable to thrombin detection and thrombin inhibitor screening in a blood clotting environment. The method comprises the following steps: reacting a polypeptide microarray chip with activated plasma or activated plasma containing a thrombin inhibitor at 36-38 DEG C for more than 40 minutes, reacting with avidin for more than 1 hour, reacting with polypeptide modified gold nanoparticles for more than 1 hours, and detecting resonant light scattering signals of the gold nanoparticles on the polypeptide microarray chip, thus realizing the detection of the thrombin activity and the screening of the thrombin inhibitor. According to the invention, the thrombin activity can be directly detected in plasma, and common plasma, hypercoagulable plasma and hypocoagulable plasma can be preliminarily distinguished; and in a blood clotting environment, the thrombin inhibitor can be screened, thus providing a foundation for the development and application of thrombin inhibitor medicaments.
Description
Technical field
The present invention relates to a peptide species micro-array chip detects thrombin activity and screening thrombin inhibitors in blood plasma method, belong to polypeptide micro-array chip technical field.
Background technology
Zymoplasm is a kind of serine protease, and it is converted into scleroproein with Fibrinogen on the one hand in coagulation pathway, promotes platelet aggregation, accelerates blood coagulation, activates again on the other hand anticoagulant mechanism, prevents thrombosis.The formation of zymoplasm is the result of each factor interaction in the whole coagulation pathway, low-level thrombin activity is with hemorrhage relevant, high-caliber thrombin activity (Thrombin Functions During Tissue Factor-Induced Blood CoaguLation relevant with Pre-thrombosis State, Blood, 2002,100,148-152; ABriefHistoricalReviewoftheWaterfall/CascadeofBloodCoagu Lation, J.Biol.Chem., 2003,278,50819-50832).
Coagulation of blood is a complex network process that is subjected to the regulation and control of the multiple factor, the change of a certain factor concentration all can the change system in the concentration of other factors.Thrombin activity detects combined effect (the In Vivo Imaging of Thrombin Activity in Experimental Thrombi with Thrombin-Sensitive Near-Infrared MolecuLar Probe that can reflect more accurately each factor in the coagulation pathway, Arterioscler.Thromb.Vasc.Biol., 2002,22,1929-1935).The zymoplasm that detects in the existing technique of analytical chemistry in the blood plasma adopts the mark-on experiment more, the zymoplasm of introducing can the change system in the concentration of other factors, thereby cause the concentration of zymoplasm itself to change, so the detection of zymoplasm is not suitable for the coagulation of blood environment in the technique of analytical chemistry yet.
Polypeptide micro-array chip technology is a kind of research Characterization of antigenic epitopes that is widely used in, the interaction of albumen and substrate, the interaction of cell content and polypeptide, and the interactional method for high-flux analysis of small-molecule substance and albumen (Microarray-Based Detection of Protein Binding and Functionality by Gold Nanoparticle Probes, Anal.Chem., 2005,77,5770-5774).But in the prior art, also do not utilize the polypeptide micro-array chip in blood plasma, to detect the method for thrombin activity and screening thrombin inhibitors.
Summary of the invention
Be not suitable for the technical problem of the screening of the detection of zymoplasm in the coagulation of blood environment and thrombin inhibitors for solving the method that detects zymoplasm in the existing analytical chemistry, the invention provides a peptide species micro-array chip detects thrombin activity and screens thrombin inhibitors in blood plasma method.
Polypeptide micro-array chip of the present invention detects the method for thrombin activity in blood plasma, may further comprise the steps:
(1) blood plasma with polypeptide micro-array chip and activation reacts more than the 40min at 36-38 ° of C, obtains reacted polypeptide micro-array chip;
Described polypeptide micro-array chip is take the polypeptide of biotin modification as substrate, the aminoacid sequence of described polypeptide is Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys, Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys, Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys, Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s or Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys, wherein, vitamin H all is modified on the terminal Methionin, D-Phe is D type phenylalanine, and other amino acid is L-type amino acid;
(2) with more than reacted polypeptide micro-array chip and avidin (avidin) the reaction 1h, with more than the peptide modified golden nanometer particle reaction 1h, obtain the polypeptide micro-array chip of golden nanometer particle mark again;
Described peptide modified golden nanometer particle is the golden nanometer particle that Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly modify, and wherein, is modified with vitamin H on the Lys;
(3) the resonant light scattering signal of golden nanometer particle on the polypeptide micro-array chip of detection golden nanometer particle mark is realized the detection to thrombin activity.
Preferably, the polypeptide dot matrix concentration of described polypeptide micro-array chip is 0.1-2mg/mL.
Preferably, the blood plasma with polypeptide micro-array chip and activation reacts 40min at 37 ° of C.
Preferably, the concentration of described avidin is more than the 3 μ M.
Preferably, the concentration of described peptide modified golden nanometer particle is more than the 3nM.
Preferably, the median size of described golden nanometer particle is 25-30nm.
Preferably, the volume of the blood plasma of described activation is 30 μ L.
Preferably, the blood plasma of described activation is to adopt the following methods preparation: add dilution buffer liquid, recombinant human tissue factor, mixture of phospholipids and calcium chloride in blood plasma.
The present invention also provides the polypeptide micro-array chip to screen the method for thrombin inhibitors in blood plasma, may further comprise the steps:
(1) blood plasma of polypeptide micro-array chip with the activation that adds thrombin inhibitors is reacted more than the 40min at 36-38 ° of C, obtain reacted polypeptide micro-array chip;
Described polypeptide micro-array chip is take the polypeptide of biotin modification as substrate, the aminoacid sequence of described polypeptide is Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys, Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys, Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys, Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s or Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys, wherein, vitamin H all is modified on the terminal Methionin, D-Phe is D type phenylalanine, and other amino acid is L-type amino acid;
(2) with more than reacted polypeptide micro-array chip and the avidin reaction 1h, with more than the peptide modified golden nanometer particle reaction 1h, obtain the polypeptide micro-array chip of golden nanometer particle mark again;
Described peptide modified golden nanometer particle is the golden nanometer particle that Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly modify, and wherein, is modified with vitamin H on the Lys;
(3) the resonant light scattering signal of golden nanometer particle on the polypeptide micro-array chip of detection golden nanometer particle mark is realized the screening to thrombin inhibitors.
Preferably, described thrombin inhibitors is argatroban, Antithrombin III or 4-(2-aminoethyl) benzene sulfonyl fluorine hydrochloride (AEBSF).
The invention has the beneficial effects as follows:
(1) the present invention prepares the polypeptide micro-array chip take the polypeptide of biotin modification as substrate, in blood plasma, detect thrombin activity and screening thrombin inhibitors, the thrombin action site of the thrombin action peptide substrate on chip that activates in the blood plasma that at first activates, make the polypeptide fragment with vitamin H be hydrolyzed, retain the polypeptide fragment of not being with vitamin H on the chip, then utilize the Avidin-Biotin reaction, adopt first avidin labeling polypeptide micro-array chip, the golden nanometer particle tagging chip of modifying with Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly again, resonant light scattering signal (the Resonance Light Scattering of golden nanometer particle on the chip of detection golden nanometer particle mark, RLS), after chip and the thrombin action, part is dissociated from chip with the polypeptide fragment of vitamin H, the resonant light scattering signal of polypeptide dot matrix weakens thereupon, by detecting the resonant light scattering signal, realize the detection of thrombin activity and the screening of thrombin inhibitors;
(2) method of the present invention can be used for the direct-detection of thrombin activity in the blood plasma, and adopts from the upstream of coagulation of blood cascade reaction and activate coagulation pathway, tentatively distinguishes common blood plasma, high blood coagulation slurry and hangs down the blood coagulation slurry;
(3) the present invention can screen the inhibitor of zymoplasm in blood plasma, can provide for the screening of inhibitor one near real blood plasma environment, for preliminary judgement is made in the impact of prediction practical application other factor pair inhibitor in the human body environment, judge more accurately the inhibition of inhibitor, realize high-throughout, micro-, fast, the screening of sensitive thrombin inhibitors, for the research of thrombin inhibitors class medicine with should be used as the basis.
Description of drawings
Fig. 1 is the lattice optics photo of the polypeptide micro-array chip of different concns polypeptide dot matrix of the present invention;
Fig. 2 is the polypeptide micro-array chip of the embodiment of the invention 1 different peptide substrates and the resonant light scattering change in signal strength after the thrombin action;
Fig. 3 is the polypeptide dot matrix concentration of polypeptide micro-array chip of the embodiment of the invention 2 and embodiment 3 and the double reciprocal curve of catalyzed by thrombin speed of response;
Fig. 4 is the canonical plotting that the polypeptide micro-array chip of the embodiment of the invention 4 detects zymoplasm;
Fig. 5 be the polypeptide micro-array chip of the embodiment of the invention 5 and the common blood plasma of activation, high blood coagulation slurry and hang down the effect of blood coagulation slurry after the resonant light scattering change in signal strength;
Fig. 6 is the resonant light scattering signal after the blood plasma effect of polypeptide micro-array chip and the activation that adds thrombin inhibitors of the embodiment of the invention 6;
Fig. 7 is the schema of Comparative Examples 1 of the present invention;
Fig. 8 is the schema of Comparative Examples 2 of the present invention;
Fig. 9 is the schema of Comparative Examples 3 of the present invention;
Figure 10 is the schema that embodiment of the invention 1-4 polypeptide micro-array chip detects zymoplasm;
Figure 11 is the schema that the embodiment of the invention 5 polypeptide micro-array chips detect plasma sample;
Figure 12 is the embodiment of the invention 6 polypeptide micro-array chips screen thrombin inhibitors in blood plasma schema.
Among the figure,
Be polypeptide,
Be the polypeptide of biotin modification,
Be avidin,
Be peptide modified golden nanometer particle.
Embodiment
The polypeptide micro-array chip detects the method for thrombin activity in blood plasma, may further comprise the steps:
(1) blood plasma with polypeptide micro-array chip and activation reacts more than the 40min at 36-38 ° of C, the thrombin action site of the thrombin action peptide substrate on chip that activates in the blood plasma that activates, make the polypeptide fragment with vitamin H be hydrolyzed, retain the polypeptide fragment of not being with vitamin H on the chip, obtain reacted polypeptide micro-array chip;
Described polypeptide micro-array chip is take the polypeptide of biotin modification as substrate, the aminoacid sequence of described polypeptide is Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys, Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys, Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys, Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s or Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys, wherein, vitamin H all is modified on the terminal Methionin, D-Phe is D type phenylalanine, and other amino acid is L-type amino acid;
(2) with more than reacted polypeptide micro-array chip and the avidin reaction 1h, with more than the peptide modified golden nanometer particle reaction 1h, obtain the polypeptide micro-array chip of golden nanometer particle mark again;
Described peptide modified golden nanometer particle is the golden nanometer particle that Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly modify, and wherein, is modified with vitamin H on the Lys;
(3) the resonant light scattering signal of golden nanometer particle on the polypeptide micro-array chip of detection golden nanometer particle mark, behind the polypeptide micro-array chip and thrombin action of step (1), part is dissociated from chip with the polypeptide fragment of vitamin H, the resonant light scattering signal of dot matrix weakens thereupon, and the mensuration by the resonant light scattering signal realizes the detection to thrombin activity.
Among the present invention, polypeptide micro-array chip take Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s (biotin) as peptide substrate and the affinity of zymoplasm are higher, the susceptibility of its detection is better, so the preferred Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s of the present invention (biotin) is the polypeptide micro-array chip of peptide substrate detects zymoplasm in blood plasma activity.
Among the present invention, in the situation of the aminoacid sequence of knowing polypeptide, the acquisition of peptide substrate is those skilled in the art's known technology, and the peptide substrate of present embodiment is synthetic by the Shanghai bio tech ltd of shining by force.
Among the present invention, employed chip does not have particular requirement, be purchased to get final product, present embodiment employing Beijing Boao Biological Co., Ltd
Polymer three-dimensional substrate D.
The preparation method of polypeptide micro-array chip of the present invention is those skilled in the art's known technology, and such as contact process, present embodiment provides a kind of its preparation process, but the invention is not restricted to this, and concrete steps are:
(1) use Beijing Boao Biological Co., Ltd to produce
SmartArrayer136 point sample system exists polypeptide solution point
Polymer three-dimensional substrate D surface;
(2) place vacuum drying oven to vacuumize said chip, 30 ° of C reaction overnight obtain reacted chip;
(3) with the chip behind 1% (w/v) bovine serum albumin capping, make the polypeptide micro-array chip.
Among the present invention, the polypeptide dot matrix concentration of polypeptide micro-array chip generally adopts 0.1-2mg/mL, but be not limited to this, the concentration of polypeptide dot matrix is larger in the polypeptide micro-array chip, the resonant light scattering signal is stronger, as shown in Figure 1, the peptide substrate that adopts the Arrayit microarray scanner to measure is the lattice optics photo of the polypeptide micro-array chip of Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s (biotin), from left to right polypeptide dot matrix concentration be followed successively by 0.2,0.3,0.35,0.5,1,1.5mg/mL.
Among the present invention, the concentration of avidin is 3 μ M.
Among the present invention, the concentration of peptide modified golden nanometer particle is 3nM, and median size is 25-30nm, preferred 30nm (the ultraviolet maximum absorption band is 528nm).
Among the present invention, the volume of the blood plasma that activates is decided by the size of reaction vessel, in the present embodiment, with the volume of the blood plasma of the activation of polypeptide micro-array chip effect be 30 μ L, can adopt the following methods preparation: in blood plasma (40 μ L), add dilution buffer liquid (40 μ L), recombinant human tissue factor, mixture of phospholipids and calcium chloride, the blood plasma that obtains activating, the concentration of recombinant human tissue factor is 5pM in the blood plasma that activates, the concentration of mixture of phospholipids is that the concentration of 4 μ M and calcium chloride is 17mM, described dilution buffer liquid is the 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES that contains 60mg/mL bovine serum albumin and 21.7mM trisodium citrate, 20mM) solution, described mixture of phospholipids are the two oleoyl phosphorylcholines of 60mol%, the two oleoyl phosphoserines of the DOPE of 20mol% and 20mol%.
When utilizing polypeptide micro-array chip of the present invention to detect zymoplasm in blood plasma, the linear detection range of zymoplasm is 2.7 * 10
-2-810nM detects and is limited to 19.7pM.
Polypeptide micro-array chip of the present invention detects the method for thrombin activity and can and hang down the blood coagulation slurry and tentatively distinguish common blood plasma, high blood coagulation slurry in blood plasma.
The polypeptide micro-array chip screens the method for thrombin inhibitors in blood plasma, may further comprise the steps:
(1) blood plasma of polypeptide micro-array chip with the activation that adds thrombin inhibitors is reacted more than the 40min at 36-38 ° of C, obtain reacted polypeptide micro-array chip;
Described polypeptide micro-array chip is take the polypeptide of biotin modification as substrate, the aminoacid sequence of described polypeptide is Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys, Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys, Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys, Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s or Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys, wherein, vitamin H all is modified on the terminal Methionin, D-Phe is D type phenylalanine, and other amino acid is L-type amino acid;
(2) with more than reacted polypeptide micro-array chip and the avidin reaction 1h, with more than the peptide modified golden nanometer particle reaction 1h, obtain the polypeptide micro-array chip of golden nanometer particle mark again;
Described peptide modified golden nanometer particle is the golden nanometer particle that Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly modify, and wherein, is modified with vitamin H on the Lys;
(3) the resonant light scattering signal of golden nanometer particle on the polypeptide micro-array chip of detection golden nanometer particle mark is realized the screening to thrombin inhibitors.
Among the present invention, thrombin inhibitors does not have particular determination, is generally argatroban, Antithrombin III or AEBSF, and the concentration of thrombin inhibitors in blood plasma is 7.5 μ M.
Among the present invention, polypeptide micro-array chip take Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s (biotin) as peptide substrate and the affinity of zymoplasm are higher, the susceptibility of its detection is better, so the preferred Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s of the present invention (biotin) screens thrombin inhibitors for the polypeptide micro-array chip of peptide substrate in blood plasma.
Among the present invention, in the situation of the aminoacid sequence of knowing polypeptide, the acquisition of peptide substrate is those skilled in the art's known technology, and the peptide substrate of present embodiment is synthetic by the Shanghai bio tech ltd of shining by force.
Among the present invention, employed chip does not have particular requirement, be purchased to get final product, present embodiment employing Beijing Boao Biological Co., Ltd
Polymer three-dimensional substrate D.
The preparation method of polypeptide micro-array chip of the present invention is those skilled in the art's known technology, and such as contact process, present embodiment provides a kind of its preparation process, but the invention is not restricted to this, and concrete steps are:
(1) use Beijing Boao Biological Co., Ltd to produce
SmartArrayer136 point sample system exists polypeptide solution point
Polymer three-dimensional substrate D surface;
(2) place vacuum drying oven to vacuumize said chip, 30 ° of C reaction overnight obtain reacted chip;
(3) with the chip behind 1% (w/v) bovine serum albumin capping, make the polypeptide micro-array chip.
Among the present invention, the polypeptide dot matrix concentration of polypeptide micro-array chip generally adopts 0.1-2mg/mL, but be not limited to this, the concentration of polypeptide dot matrix is larger in the polypeptide micro-array chip, the resonant light scattering signal is stronger, as shown in Figure 1, the peptide substrate that adopts the Arrayit microarray scanner to measure is the lattice optics photo of the polypeptide micro-array chip of Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s (biotin), from left to right polypeptide dot matrix concentration be followed successively by 0.2,0.3,0.35,0.5,1,1.5mg/mL.
Among the present invention, the concentration of avidin is 3 μ M.
Among the present invention, the concentration of peptide modified golden nanometer particle is 3nM, and median size is 25-30nm, preferred 30nm (the ultraviolet maximum absorption band is 528nm).
Comparative Examples 1
In conjunction with Fig. 7 Comparative Examples 1 is described
(1) be 0.5mg/mL with polypeptide dot matrix concentration, peptide substrate is respectively:
Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys(biotin)；
Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys(biotin)；
Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；
Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；
Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys(biotin)；
Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys(biotin)；
Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；
The polypeptide micro-array chip of Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys (biotin), respectively with the damping fluid reaction 1h that does not contain zymoplasm, obtain reacted polypeptide micro-array chip, the pH value of described damping fluid is 7.35, contain the 20mM4-hydroxyethyl piperazine ethanesulfonic acid, 0.14mM sodium-chlor and 2mM calcium chloride;
(2) with reacted polypeptide micro-array chip respectively successively with 3 μ M avidins, the peptide modified golden nanometer particle of 3nM respectively reacts 1h, obtains the polypeptide micro-array chip of golden nanometer particle mark;
(3) the polypeptide micro-array chip of golden nanometer particle mark is measured golden nanometer particle resonant light scattering signal with the Arrayit microarray scanner.
The result shows, the resonant light scattering strength of signal of the polypeptide micro-array chip of Comparative Examples 1 golden nanometer particle mark is constant, and Fig. 7 is the schema of Comparative Examples 1 of the present invention.
Comparative Examples 2
In conjunction with Fig. 8 Comparative Examples 2 is described
(1) be 0.5mg/mL with polypeptide dot matrix concentration, peptide substrate is the polypeptide micro-array chip of Cys-Ala-Leu-Asn-Asn, and the zymoplasm reaction 1h with 27nM obtains reacted polypeptide micro-array chip;
(2) with reacted polypeptide micro-array chip successively with 3 μ M avidins, the peptide modified golden nanometer particle of 3nM respectively reacts 1h, obtains the polypeptide micro-array chip of golden nanometer particle mark;
(3) the polypeptide micro-array chip of golden nanometer particle mark is measured golden nanometer particle resonant light scattering signal with the Arrayit microarray scanner.
The result shows, the polypeptide micro-array chip of the golden nanometer particle mark of Comparative Examples 2 can not produce the resonant light scattering signal, and Fig. 8 is the schema of Comparative Examples 2 of the present invention.
In conjunction with the generation of generation, variation and the test-results of resonant light scattering signal among the 2 explanation the present invention of Comparative Examples 1 and Comparative Examples, all be that the effect by zymoplasm causes, get rid of the possibility that other factors produces signal intensity and test-results.
Comparative Examples 3
In conjunction with Fig. 9 Comparative Examples 3 is described
(1) be 0.5mg/mL with polypeptide dot matrix concentration, peptide substrate is the polypeptide micro-array chip of Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s (biotin), respectively with the unactivated common blood plasma of 30 μ L, the unactivated high blood coagulation slurry of 30 μ L, the unactivated low blood coagulation slurry of 30uL obtains reacted polypeptide micro-array chip at 37 ° of C reaction 40min;
Described common blood plasma is the fresh frozen plasma of Healthy People, described high blood coagulation slurry is untreated spontaneous deep venous thrombosis (Deep vein thrombosis, DVT) or pulmonary infarction (PuLmonary embolism, PE) patient's fresh frozen plasma, described low blood coagulation slurry is the fresh frozen plasma of the Healthy People of adding 7.5 μ M thrombin inhibitorss (Antithrombin III);
(2) with reacted polypeptide micro-array chip respectively successively with 3 μ M avidins, the peptide modified golden nanometer particle of 3nM respectively reacts 1h, obtains the polypeptide micro-array chip of golden nanometer particle mark;
(3) the polypeptide micro-array chip of golden nanometer particle mark is measured golden nanometer particle resonant light scattering signal with the Arrayit microarray scanner.
Fig. 9 is the schema of Comparative Examples 3 of the present invention.
In conjunction with Fig. 2 and Figure 10 embodiment 1 is described
The polypeptide micro-array chip detects thrombin activity:
(1) with the polypeptide micro-array chip of 8 peptide species substrates, polypeptide dot matrix concentration is 0.5mg/mL, and 8 peptide species substrates are respectively:
Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys(biotin)；
Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys(biotin),；
Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；
Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；
Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys(biotin)；
Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys(biotin)；
Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；
The polypeptide micro-array chip of Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys (biotin) reacts 1h with the 27nM zymoplasm respectively, obtains reacted polypeptide micro-array chip;
(2) with reacted polypeptide micro-array chip respectively successively with 3 μ M avidins, the peptide modified golden nanometer particle of 3nM respectively reacts 1h, obtains the polypeptide micro-array chip of golden nanometer particle mark;
(3) the polypeptide micro-array chip of golden nanometer particle mark is measured golden nanometer particle resonant light scattering signal with the Arrayit microarray scanner.
The golden nanometer particle resonant light scattering signal of the 8 peptide species dot matrix that embodiment 2 is obtained respectively with the golden nanometer particle resonant light scattering signal of the same peptide species dot matrix of Comparative Examples 1 relatively, resonant light scattering change in signal strength result is denoted as respectively S01, S02, S03, S04, S05, S06, S07, S08 makes the polypeptide micro-array chip of different peptide substrates and the resonant light scattering change in signal strength after the thrombin action, as shown in Figure 2, as can be seen from Figure 2,8 peptide species micro-array chips of the present invention can both with thrombin action, and the variation by the resonant light scattering strength of signal shows, wherein, zymoplasm is maximum to the functioning efficiency of S07 polypeptide, so that substrate is the avidity of the chip of Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s (biotin) and zymoplasm is higher.
In conjunction with Fig. 3 and Figure 10 embodiment 2 is described
The polypeptide micro-array chip detects thrombin activity:
(1) polypeptide dot matrix concentration is respectively 0.2,0.3,0.35,0.5,1,1.5mg/mL, peptide substrate is that the polypeptide micro-array chip of Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s (biotin) reacts 1h with the 810nM zymoplasm respectively, obtains reacted polypeptide micro-array chip;
(2) with reacted polypeptide micro-array chip respectively successively with 3 μ M avidins, the peptide modified golden nanometer particle of 3nM respectively reacts 1h, obtains the polypeptide micro-array chip of golden nanometer particle mark;
(3) the polypeptide micro-array chip of golden nanometer particle mark is measured golden nanometer particle resonant light scattering signal with the Arrayit microarray scanner.
Embodiment 3
In conjunction with Fig. 3 and Figure 10 embodiment 3 is described
The polypeptide micro-array chip detects thrombin activity:
(1) polypeptide dot matrix concentration is respectively 0.1,0.15,0.2,0.3,1,2mg/mL, peptide substrate is that the polypeptide micro-array chip of Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys (biotin) reacts 1h with the 810nM zymoplasm respectively, obtains reacted polypeptide micro-array chip;
(2) with reacted polypeptide micro-array chip respectively successively with 3 μ M avidins, the peptide modified golden nanometer particle of 3nM respectively reacts 1h, obtains the polypeptide micro-array chip of golden nanometer particle mark;
(3) the polypeptide micro-array chip of golden nanometer particle mark is measured golden nanometer particle resonant light scattering signal with the Arrayit microarray scanner.
Fig. 3 is the polypeptide dot matrix concentration of polypeptide micro-array chip of embodiment 2 and the double reciprocal curve of catalyzed by thrombin speed of response a), Fig. 3 b) the peptide substrate sequence that provides take proteolytic enzyme database (MEROPS) for embodiment 3 is the double reciprocal curve of polypeptide dot matrix concentration and the catalyzed by thrombin speed of response of the chip of substrate, as can be seen from Figure 3, the avidity of the polypeptide of the polypeptide of embodiment 2 and embodiment 3 and zymoplasm is better.
In conjunction with Fig. 4 and Figure 10 embodiment 4 is described
The polypeptide micro-array chip detects thrombin activity:
(1) be 0.5mg/mL with polypeptide dot matrix concentration, peptide substrate is that the polypeptide micro-array chip of Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s (biotin) is 0.0135,0.027,0.135,1.35,13.5,135,405,810 with concentration respectively, the zymoplasm of 1350nM reaction 1h, obtains reacted polypeptide micro-array chip;
(2) with reacted polypeptide micro-array chip respectively successively with 3 μ M avidins, the peptide modified golden nanometer particle of 3nM respectively reacts 1h, obtains the polypeptide micro-array chip of golden nanometer particle mark;
(3) the polypeptide micro-array chip of golden nanometer particle mark is measured golden nanometer particle resonant light scattering signal with the Arrayit microarray scanner.
Fig. 4 is the canonical plotting that the embodiment of the invention 4 polypeptide micro-array chips detect zymoplasm; As can be seen from Figure 4, the linearity range of zymoplasm detection of the present invention is 2.7 * 10
-2-810nM detects and is limited to 19.7pM.
Figure 10 is the schema that embodiment of the invention 1-4 polypeptide micro-array chip detects zymoplasm.
Embodiment 5
In conjunction with Fig. 5 and Figure 11 embodiment 5 is described
The polypeptide micro-array chip detects thrombin activity in blood plasma:
(1) be 0.5mg/mL with polypeptide dot matrix concentration, peptide substrate is that the polypeptide micro-array chip of Cys-Ala-Glu-Gly-Gly-D-Phe-ProArg-Ser-Phe-Arg-Val-Val-Lys (biotin) is starched at 37 ° of C reaction 40min with common blood plasma, the high blood coagulation slurry of 4 groups of 30uL activation and the low blood coagulation of 7 groups of 30uL activation that 13 groups of 30uL activate respectively, obtains reacted polypeptide micro-array chip;
Described common blood plasma is the fresh frozen plasma of Healthy People, described high blood coagulation slurry is untreated spontaneous deep venous thrombosis (Deep vein thrombosis, DVT) or pulmonary infarction (PuLmonary embolism, PE) patient's fresh frozen plasma, described low blood coagulation slurry is the fresh frozen plasma of the Healthy People of adding 7.5 μ M thrombin inhibitorss (Antithrombin III);
(2) with reacted polypeptide micro-array chip respectively successively with 3 μ M avidins, the peptide modified golden nanometer particle reaction 1h of 3nM obtains the polypeptide micro-array chip of golden nanometer particle mark;
(3) the polypeptide micro-array chip of golden nanometer particle mark is measured golden nanometer particle resonant light scattering signal with the Arrayit microarray scanner.
Fig. 5 is that the polypeptide micro-array chip of the embodiment of the invention 5 preparation detects the common blood plasma that activates, high blood coagulation slurry and hangs down thrombin activity figure in the blood coagulation slurry, as can be seen from Figure 5, when golden nanometer particle resonant light scattering change amount signal 50.2% when following, blood is low blood coagulation slurry, when golden nanometer particle resonant light scattering change amount signal 73.2% when above, blood is high blood coagulation slurry.
Figure 11 is the embodiment of the invention 5 polypeptide micro-array chips detect thrombin activity in blood plasma schema.
In conjunction with Fig. 6 and Figure 12 embodiment 6 is described
The polypeptide micro-array chip screens thrombin inhibitors in blood plasma:
(1) be 0.5mg/mL with polypeptide dot matrix concentration, peptide substrate be Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s (biotin) the polypeptide micro-array chip respectively with the common blood plasma that activates, add the common blood plasma of the activation of argatroban, the common blood plasma that adds the activation of Antithrombin III, the common blood plasma that adds the activation of AEBSF, with unactivated common blood plasma reaction 40min, obtain reacted polypeptide micro-array chip, the final concentration of argatroban, Antithrombin III and AEBSF is 7.5 μ M;
(2) with reacted polypeptide micro-array chip respectively successively with 3 μ M avidins, the peptide modified golden nanometer particle of 3nM respectively reacts 1h, obtains the polypeptide micro-array chip of golden nanometer particle mark;
(3) the polypeptide micro-array chip of golden nanometer particle mark is measured golden nanometer particle resonant light scattering signal with the Arrayit microarray scanner.
Fig. 6 is the resonant light scattering signal after the blood plasma effect of polypeptide micro-array chip and the activation that adds thrombin inhibitors of the embodiment of the invention 6; As can be seen from Figure 6, argatroban and Antithrombin III have the ability of higher Trombin inhibiting, and the present invention can directly screen the inhibitor of zymoplasm in blood plasma.
Figure 12 is the embodiment of the invention 6 polypeptide micro-array chips screen thrombin inhibitors in blood plasma schema.
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.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 carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (10)
1. the polypeptide micro-array chip detects the method for thrombin activity in blood plasma, it is characterized in that, may further comprise the steps:
(1) blood plasma with polypeptide micro-array chip and activation reacts more than the 40min at 36-38 ° of C, obtains reacted polypeptide micro-array chip;
Described polypeptide micro-array chip is take the polypeptide of biotin modification as substrate, the aminoacid sequence of described polypeptide is Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys, Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys, Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys, Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s or Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys, wherein, vitamin H all is modified on the terminal Methionin, D-Phe is D type phenylalanine, and other amino acid is L-type amino acid;
(2) with more than reacted polypeptide micro-array chip and the avidin reaction 1h, with more than the peptide modified golden nanometer particle reaction 1h, obtain the polypeptide micro-array chip of golden nanometer particle mark again;
Described peptide modified golden nanometer particle is the golden nanometer particle that Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly modify, and wherein, is modified with vitamin H on the Lys;
(3) the resonant light scattering signal of golden nanometer particle on the polypeptide micro-array chip of detection golden nanometer particle mark is realized the detection to thrombin activity.
2. polypeptide micro-array chip according to claim 1 detects the method for thrombin activity in blood plasma, it is characterized in that, the polypeptide dot matrix concentration of described polypeptide micro-array chip is 0.1-2mg/mL.
3. polypeptide micro-array chip according to claim 1 detects the method for thrombin activity in blood plasma, it is characterized in that, the blood plasma of described polypeptide micro-array chip and activation is at 37 ° of C reaction 40min.
4. polypeptide micro-array chip according to claim 1 detects the method for thrombin activity in blood plasma, it is characterized in that, the concentration of described avidin is more than the 3 μ M.
5. polypeptide micro-array chip according to claim 1 detects the method for thrombin activity in blood plasma, it is characterized in that, the concentration of described peptide modified golden nanometer particle is more than the 3nM.
6. polypeptide micro-array chip according to claim 1 detects the method for thrombin activity in blood plasma, it is characterized in that, the median size of described golden nanometer particle is 25-30nm.
7. polypeptide micro-array chip according to claim 1 detects the method for thrombin activity in blood plasma, it is characterized in that, the volume of the blood plasma of described activation is 30 μ L.
8. polypeptide micro-array chip according to claim 1 detects the method for thrombin activity in blood plasma, it is characterized in that, the blood plasma of described activation is to adopt the following methods preparation: add dilution buffer liquid, recombinant human tissue factor, mixture of phospholipids and calcium chloride in blood plasma.
9. the polypeptide micro-array chip screens the method for thrombin inhibitors in blood plasma, it is characterized in that, may further comprise the steps:
(1) blood plasma of polypeptide micro-array chip with the activation that adds thrombin inhibitors is reacted more than the 40min at 36-38 ° of C, obtain reacted polypeptide micro-array chip;
Described polypeptide micro-array chip is take the polypeptide of biotin modification as substrate, the aminoacid sequence of described polypeptide is Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys, Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys, Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys, Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Ly s or Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys, wherein, vitamin H all is modified on the terminal Methionin, D-Phe is D type phenylalanine, and other amino acid is L-type amino acid;
(2) with more than reacted polypeptide micro-array chip and the avidin reaction 1h, with more than the peptide modified golden nanometer particle reaction 1h, obtain the polypeptide micro-array chip of golden nanometer particle mark again;
Described peptide modified golden nanometer particle is the golden nanometer particle that Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly modify, and wherein, is modified with vitamin H on the Lys;
(3) the resonant light scattering signal of golden nanometer particle on the polypeptide micro-array chip of detection golden nanometer particle mark is realized the screening to thrombin inhibitors.
10. polypeptide micro-array chip according to claim 9 screens the method for thrombin inhibitors in blood plasma, it is characterized in that, described thrombin inhibitors is argatroban, Antithrombin III or 4-(2-aminoethyl) benzene sulfonyl fluorine hydrochloride.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310282186.XA CN103361398B (en) | 2013-07-05 | 2013-07-05 | Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310282186.XA CN103361398B (en) | 2013-07-05 | 2013-07-05 | Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103361398A true CN103361398A (en) | 2013-10-23 |
| CN103361398B CN103361398B (en) | 2015-05-20 |
Family
ID=49363654
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310282186.XA Active CN103361398B (en) | 2013-07-05 | 2013-07-05 | Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103361398B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105884888A (en) * | 2016-04-14 | 2016-08-24 | 上海大学 | Artificial antibody construction method based on gold nanoparticles |
| CN114231594A (en) * | 2021-09-29 | 2022-03-25 | 深圳市赛尔生物技术有限公司 | Method for detecting thrombin activity and screening thrombin inhibitor in plasma by polypeptide microarray chip |
-
2013
- 2013-07-05 CN CN201310282186.XA patent/CN103361398B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| GAO JQ ET AL: "Microarray-Based Study of Carbohydrate-Protein Binding by Gold Nanoparticle Probes", 《ANAL CHEM》 * |
| GAO JQ ET AL: "Screening Lectin-Binding Specificity of Bacterium by Lectin Microarray with Gold Nanoparticle Probes", 《ANAL CHEM》 * |
| LI T ET AL: "Screening Kinase Inhibitors with a Microarray-Based Fluorescent and Resonance Light Scattering Assay", 《ANAL CHEM》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105884888A (en) * | 2016-04-14 | 2016-08-24 | 上海大学 | Artificial antibody construction method based on gold nanoparticles |
| CN114231594A (en) * | 2021-09-29 | 2022-03-25 | 深圳市赛尔生物技术有限公司 | Method for detecting thrombin activity and screening thrombin inhibitor in plasma by polypeptide microarray chip |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103361398B (en) | 2015-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Garvey et al. | The detection of fibrinogen/fibrin degradation products by means of a new antibody-coated latex particle | |
| US10935559B2 (en) | Methodologies and reagents for detecting fibrinolysis and hyperfibrinolysis | |
| CN105403693B (en) | A kind of preparation method of magnetic microparticle chemiluminescence reagent | |
| JP5514101B2 (en) | Method for measuring inhibition of platelet aggregation by thrombin receptor antagonist | |
| Midura et al. | Microparticles impact coagulation after traumatic brain injury | |
| JP6877340B2 (en) | Confirmation of new pathological conditions using viscoelastic analysis in the presence of thrombolytic agents | |
| Parsi et al. | In vitro effects of detergent sclerosants on coagulation, platelets and microparticles | |
| JP5437660B2 (en) | Coagulogen raw material, method for producing the same, method and apparatus for measuring biologically active substances derived from organisms using the same | |
| CN104198731B (en) | A kind of c reactive protein half-quantitative detection reagent and apply the test paper of this reagent | |
| CN103454193A (en) | Immunoturbidimetric kit for detecting lipoprotein (a) and preparation method thereof | |
| Pretorius et al. | Ultrastructural comparison of the morphology of three different platelet and fibrin fiber preparations | |
| CN102692511B (en) | Kit (Developing substrate method) for testing protein C (PC) | |
| CN103361398B (en) | Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip | |
| CN103969453B (en) | Alpha-fetoprotein autoantibody individually or combines the application monitoring mark as diagnosing tumor, curative effect evaluation and recurrence with alpha-fetoprotein | |
| EP1313877B1 (en) | Assay for soluble fibrin | |
| Tamura et al. | A new sensitive method for determining endotoxin in whole blood | |
| CN205301329U (en) | D - dimer and fibrinogen ELISA kit | |
| Obata et al. | Early detection of the Limulus amebocyte lysate reaction evoked by endotoxins | |
| Zhang et al. | A novel insight into mechanism of derangement of coagulation balance: interactions of quantum dots with coagulation-related proteins | |
| Marx et al. | The activation peptide of thrombin‐activatable fibrinolysis inhibitor: a role in activity and stability of the enzyme? | |
| CN104698159A (en) | Method for detecting endotoxin content | |
| CN105440127B (en) | It is a kind of using human plasma Cohn components III as the preparation method of the FEIBA of raw material | |
| JP2020523577A (en) | Novel method for preserving serum protein activity | |
| JP3712989B2 (en) | Method for producing freeze-dried plasma | |
| Min et al. | A peptide microarray-based fluorescent and resonance light scattering assay for screening thrombin inhibitor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220729 Address after: No. 5218, Longhu Road, Changchun City, Jilin Province, 130000 Patentee after: Zhongke Yinghua (Changchun) Technology Co.,Ltd. Address before: 130022 5625 people's street, Chaoyang District, Changchun, Jilin. Patentee before: CHANGCHUN INSTITUTE OF APPLIED CHEMISTRY CHINESE ACADEMY OF SCIENCES |
|
| TR01 | Transfer of patent right |