CN114133445B

CN114133445B - Preparation method of fibrinogen degradation fragment

Info

Publication number: CN114133445B
Application number: CN202111459024.XA
Authority: CN
Inventors: 丁重辉; 李博华; 王晓
Original assignee: Beijing Succeeder Technology Inc
Current assignee: Beijing Succeeder Technology Inc
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2023-11-17
Anticipated expiration: 2041-12-01
Also published as: CN114133445A

Abstract

The application relates to a fibrinogen degradation technology, and discloses a preparation method of fibrinogen degradation fragments, which is characterized in that a plasmin solution is added into a fibrinogen solution for degradation, the plasmin is required to be degraded in a degradation process according to the matching of 6IU of each 1mg of fibrinogen, the degradation time is 30min, a mixture of fragments X, Y, D and E is obtained, and a fibrinogen degradation product target fragment is obtained through separation and purification. The method provided by the application can be used for accurately preparing target fragments, and each degradation product peptide fragment prepared by the method has the characteristics of high purity and high concentration, can be used for preparing FDP calibrator and subsequent antibody screening antigen, and has positive significance in the field of clinical detection.

Description

Preparation method of fibrinogen degradation fragment

Technical Field

The application relates to fibrinogen degradation technology, in particular to a preparation method of fibrinogen degradation fragments.

Background

The fibrinolysis system (fibrinolysis system) is called as fibrinolysis system for short, is the most important anticoagulation system for human body, and refers to a system that plasmin is converted into plasmin under the action of a plasmin activator, and then plasmin degrades fibrin (protogen) and other proteins.

The fibrinolytic system plays an important role in maintaining normal permeability of the vessel wall, maintaining the flowing state of blood and tissue repair. When fibrin and fibrinogen are in the presence of fibrin enzymes and Ca ²⁺ Under the action of the chain alpha of different fibrin molecules, the fibrin is converted into insoluble fibrin polymer to form fibrinWhen white blood cells are overlapped in a staggered manner and the original sol-like blood is converted into gel-like blood clots, a fibrinolytic system activates plasminogen through an internal activation or external activation way, and fibrin and fibrinogen are hydrolyzed into various dimers, polymers and complexes, which are collectively called fibrin/Fibrinogen Degradation Products (FDPs). At the same time, FDP can inhibit fibrin formation, has antithrombin effect, inhibits platelet adhesion aggregation and release, and FDP can not be coagulated any more.

Wherein, the internal activation is mainly started by related factors of an endogenous coagulation system, and the activated factors XIIa, xia, HMWK and kallikrein participate in the activation process, and the pathway is the theoretical basis of secondary fibrinolysis; the external activation pathway is mainly a process of converting plasminogen into plasmin by tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). At the same time, t-PA and u-PA are inhibited by a plasminogen activator inhibitor (PAI-1/2/3), and fibrinolytic activity is regulated by activation and inhibition, which is the theoretical basis of primary fibrinolysis. Thus, FDP reflects mainly fibrinolysis function, and primary or secondary hyperfibrinolysis can be identified by detecting FDP content in plasma. Clinically FDP is often used as a reference index for a variety of thrombotic diseases and is listed as one of the conventional indices for laboratory diagnosis of disseminated intravascular coagulation.

FDP calibrator is needed to be detected along with the FDP measurement process, so that the accuracy of measurement results is ensured. The existing calibrator adopts D-dimer as a degradation product of fibrin, is the simplest degradation product of fibrin, can be used as a calibrator for FDP detection, and is already applied to clinic. However, the prior art has no description about the calibrator related to the fibrinogen degradation products, and the main reasons are that the fibrinogen degradation product peptide fragments comprise a plurality of peptide fragments, the degradation process is difficult to control to obtain the expected target peptide fragments, the molecular weight of the fibrinogen degradation product peptide fragments is smaller than that of the D-dimer, the difference between the product fragments is not large, and the purification and separation are difficult to carry out. At present, the technical content about preparation of fibrinogen degradation fragments X, Y, D and E is not seen, so that the preparation method of the fibrinogen degradation product fragments is provided, the defect that FDP detection is not complete clinically can be effectively overcome, and the method has positive significance for diagnosing and detecting early diseases.

Disclosure of Invention

Aiming at the defects in the prior art, the embodiment of the application provides a preparation method of fibrinogen degradation fragments, fibrinogen degradation product fragments prepared by the method, and application of the fibrinogen degradation product fragments as calibrator, immunogen or screening antigen.

In one aspect of the application, a preparation method of fibrinogen degradation fragments is provided, which is realized by the following technical scheme:

a fibrinogen degradation fragment is prepared by adding plasmin solution into fibrinogen solution for degradation, and separating and purifying to obtain fibrinogen target fragment, wherein the degradation fragment consists of an X fragment, a Y fragment, a D fragment and an E fragment.

In the above technical scheme, the fibrinogen degradation fragments X, Y, D, E are respectively formed gradually, and the first step is to hydrolyze lysine-arginine bonds on a fibrinogen peptide chain under the action of plasmin so as to obtain an X fragment; the second step is that the X segment is further degraded into a D segment and a Y segment, wherein the D segment is equivalent to the C-terminal main body of the fibrin monomer; the third step is that the Y segment comprises a D segment and an E segment, the Y segment is further degraded into the D segment and the E segment, and the E segment is equivalent to the middle main body part of the fibrin monomer and comprises a disulfide bond structure.

The molecular weight of the X fragment prepared by the degradation method is 250kd, the molecular weight of the Y fragment is 150kd, the molecular weight of the D fragment is 100kd and the molecular weight of the E fragment is 50kd.

In the degradation method, each 1mg of fibrinogen is required to be matched with 6IU of plasmin for degradation, and the degradation time is 30min.

Because the fibrinogen degradation products not only comprise fragments X, Y, D, E, but also comprise fragments X ', Y', A, B, C, H and the like, the plasmin enzyme activity required by the fibrinogen degradation is specifically required in the scheme of the application, and the fibrinogen degradation products are ensured to generate fragments X, Y, D and E by matching quantitative plasmin with the fibrinogen and controlling the degradation time, thereby providing a pre-technical condition for separating and purifying the fragment X, Y, D, E.

Preferably, the fibrinogen solution concentration is 1mg/ml and the plasmin solution concentration is 0.3IU/ml.

The degradation process of the preparation method also comprises the steps of stopping fibrinolysis reaction by aprotinin to obtain an FDP mixture, and further separating and purifying to obtain the target fibrinogen degradation fragment.

The aprotinin is adopted as an anti-fibrinolytic solvent, and based on the direct inhibition effect on the over-activated plasmin, the protection of the substrate fibrinogen is realized, the degradation of the fibrinogen by the plasmin is avoided, and compared with the synthetic anti-fibrinolytic solvent, the V factor and the VIII factor in blood plasma and the alpha 2-globulin in blood serum can be protected. Meanwhile, the aprotinin is utilized to terminate fibrinolysis reaction, so that peptide fragments in fibrinogen degradation products are fragments X, Y, D and E, the generation of peptide fragments with other sizes is avoided, the purity and concentration of each target peptide fragment are improved, and the method has positive significance for being applied to the field of clinical detection.

The fibrinolysis reaction was stopped with 0.05TIU of the aprotinin per 1mg of the fibrinogen solution.

Preferably, the aprotinin solution has a concentration of 5TIU/mL.

The separation and purification are carried out by polyacrylamide gel electrophoresis (SDS-PAGE) and protein gel recovery methods, and gel filtration chromatography.

In the fibrinogen degradation mixture obtained by the degradation method, the molecular weights of fragments X, Y, D and E are 250kd, 150kd, 100kd and 50kd in sequence, the difference between the molecular weights is not large, the fragments are difficult to separate by adopting a traditional single separation and purification method, and the defects of low purity and low concentration of the peptide fragments obtained by separation exist, so that the fragments are difficult to be used for clinical detection in reagent application. According to the technical scheme, a polyacrylamide gel electrophoresis (SDS-PAGE) method, a protein gel recovery method and a purification and separation strategy of a gel filtration chromatography method are adopted, so that fragments X, Y, D and E with high concentration and high purity can be obtained through separate separation and purification, technical support is provided for preparing FDP calibrator, and the accuracy of clinical detection is ensured.

The polyacrylamide gel electrophoresis (SDS-PAGE) and protein gel recovery method are used for separating and purifying the X fragment, the Y fragment and the D fragment; the gel filtration chromatography is used for separating and purifying the E fragment.

And the purification of the X fragment, the Y fragment and the D fragment is realized by separating different protein molecules with charges under the traction of an electric field by utilizing protein electrophoresis SDS-PAGE, and then recovering and purifying the corresponding target proteins from the electrophoresis gel by utilizing a protein recovery kit.

The gel filtration chromatography uses differences in protein molecular weight or molecular shape to separate the protein of interest from the mixture. When the degraded mixed fragment mixture passes through a gel filtration chromatography column containing packed particles, large protein molecules cannot enter the gel particles and are rapidly eluted; smaller protein molecules can enter the gel particles, the retention time of the proteins entering the gel is different, the larger the molecular weight is, the earlier the outflow time is, the smaller the molecules are, the later the elution is, and finally the proteins with different molecular sizes are separated.

Preferably, in the actual separation and purification process, the SDS-PAGE and protein gel recovery method are adopted to prepare an X fragment, a Y fragment and a D fragment, and then the gel filtration chromatography is used to prepare an E fragment; the X fragment, Y fragment and D fragment can be prepared by SDS-PAGE and protein gel recovery, and the E fragment can be prepared by gel filtration chromatography.

Preferably, the polyacrylamide gel electrophoresis is carried out by adopting 8% of separation gel and 5% of concentrated gel, wherein the 8% of separation gel is electrophoresed at 120V voltage, and the 5% of concentrated gel is electrophoresed at 80V voltage.

Preferably, the recovered X fragment, Y fragment, D fragment and E fragment are subjected to concentration test by using BCA protein concentration kit.

In another aspect of the application, fibrinogen-degrading fragments X, Y, D and E prepared by the above methods are also within the scope of the application.

Compared with the prior art, the X fragment, the Y fragment, the D fragment and the E fragment prepared by the method have the characteristics of high protein purity and high concentration.

In a third aspect of the application, the X, Y, D and E fragments can be used to prepare FDP calibrator, as well as immunogens and screeners, to immunize against antibodies.

Preferably, an FDP calibrator is said fibrinogen degrading fragment.

The beneficial effects of the application are as follows:

1) By controlling the concentration of fibrinogen and plasmin and the degradation reaction time, the degradation of other peptide molecules is effectively avoided, and the mixed degradation products of the X fragment, the Y fragment, the D fragment and the E fragment with high purity and high concentration are obtained, thereby providing a precondition for the subsequent purification and separation.

2) The X fragment, the Y fragment and the D fragment are prepared by separating and purifying by SDS-PAGE and a protein gel recovery method, the E fragment is prepared by separating and purifying by a gel filtration chromatography, and under the condition that the molecular weight of peptide fragments is small and the difference is not large, the separation and purification of fragments X, Y, D and E are realized, and the technical support is provided for preparing degradation fragments by adopting fibrinogen.

3) The prepared peptide fragments of the degradation products have the characteristics of high purity and high concentration, and can be used for preparing FDP calibrator and subsequent antibody screening antigen.

Drawings

FIG. 1 is a diagram of fragment X, Y and D electrophoreses of the degradation product of example 1 of the present application;

FIG. 2 is an elution profile of fragment E in the degradation product of example 1 of the present application;

FIG. 3 is an electrophoretogram of fragment E in the degradation product of example 1 of the present application;

FIG. 4 is an electrophoretogram of the degradation product of comparative example 1 of the present application.

Detailed Description

The following description of the present application will be made more complete and clear in view of the detailed description of the application, which is to be taken in conjunction with the accompanying drawings that illustrate only some, but not all, of the embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

As an embodiment of the present application, there is provided a method for preparing fibrinogen degradation fragments X, Y, D, E, wherein a fibrinogen degradation product mixture is obtained by adding a plasmin solution to fibrinogen solution for degradation, and each fragment of fibrinogen degradation product is prepared by separation and purification using a purification technique.

Preferably, fibrinogen as a raw material may be commercially available, may be prepared from the amino acid sequence of fibrinogen by genetic engineering techniques, or may be prepared directly from plasma by a method for preparing fibrinogen.

Preferably, the plasmin may be plasmin of other different species, such as bovine plasmin.

In the degradation step, it is necessary to carry out degradation subject to matching of 6IU of said plasmin per 1mg of said fibrinogen, and the degradation time is 30min.

Preferably, a plasmin solution with a concentration of 150. Mu.g/ml is prepared by adopting plasmin with an enzyme activity of 2IU per 1mg, and 20. Mu.l is matched with 1ml of fibrinogen solution with a concentration of 1mg/ml for degradation reaction.

Preferably, the degradation reaction is carried out at 37 ℃.

When the degradation reaction is carried out, the reaction process is preferably stopped, and the degradation reaction time is controlled to be 30 minutes. The use of an anti-fibrinolytic solvent terminates the reaction process based on direct inhibition of the over-activated plasmin. There is no particular requirement for the selection of the anti-fibrotic solvent, aprotinin or synthetic anti-fibrotic solvents, or other methods that terminate the degradation reaction process may be employed.

Preferably, in the technical scheme of the application, the reaction is stopped by adding aprotinin.

Preferably, 1ml of said fibrinogen solution matches 0.05TIU of said aprotinin during the termination of the fibrinolysis reaction. For example, the aprotinin solution concentration is 1mg/mL, the enzyme activity per 1mg of aprotinin is 5TIU, and a matching of 10. Mu.l of aprotinin solution per 1mL of fibrinogen solution is required to terminate degradation.

After the degradation reaction is terminated, a mixture of fragments X, Y, D and E is obtained, and some embodiments of the application can be separated and purified by polyacrylamide gel electrophoresis (SDS-PAGE) and protein gel recovery, and gel filtration chromatography. Wherein polyacrylamide gel electrophoresis (SDS-PAGE) and protein gel recovery are used to prepare the X, Y and D fragments; gel filtration chromatography was used to prepare the E fragment.

Preferably, the SDS-PAGE gel kit is performed as follows:

1) Distilled water or deionized water (10 mL of water is added to each gram of the gel coagulant) is added into the gel coagulant dry powder to prepare 10% solution (namely 10% AP solution), and the solution is frozen at-20 ℃ after being split into small volumes and melted for use in gel preparation.

2) SDS-PAGE gels were prepared at various concentrations as required for the experiments according to the formulation of Table 1:

TABLE 1 SDS-PAGE gels at different concentrations

3) The prepared SDS-PAGE gel was stored at 4 ℃.

4) Electrophoresis is carried out, 80V voltage is used for 5% of concentrated gel, 120-130V voltage is used for 8% of separation gel, dyeing is carried out for 30min after electrophoresis for 1h, and decolorization is observed.

Preferably, the protein gel recovery kit operates as follows:

1) Cutting part of the gel containing the target strips by using a scalpel, placing the gel into a 1.5mL centrifuge tube, decoloring the protein strips in the gel to be nearly colorless by using corresponding elimination liquid, centrifuging at the room temperature of 12000rpm for 5min, discarding the supernatant, and reserving the gel.

2) The colloid in the centrifuge tube was ground to fine pieces with a grinding rod, 400. Mu.l of solution A was added, and the mixture was shaken overnight (16-18 hours) on a decolorizing shaker at room temperature, during which time it was removed and occasionally shaken several times.

3) Centrifuge at 12000rpm for 15min at room temperature, transfer supernatant to another 2mL centrifuge tube, add 2mL of pre-chilled solution B, mix well, and place at 4deg.C for 30min.

4) Centrifuge at 12000rpm for 15min, remove supernatant, place centrifuge tube in fume hood, and evaporate residual liquid.

Preferably, the micro BCA protein concentration assay kit is operated as follows:

1) Gradient diluted Bovine Serum Albumin (BSA) standard

The BSA standard at a given concentration of 2mg/mL was diluted to different concentrations using buffer according to the formulation of Table 2:

TABLE 2 preparation of BSA standards at different concentrations

2) Preparing a Micro BCA test working solution: test solutions were prepared according to the amounts of standard and sample.

Preparing a reagent MD: 4volume MC+100volume MB

Preparing a test working solution WR: 1volume MA+1volume MD

Fully and uniformly mixing for later use.

And adding the protein sample and the test working solution WR according to the proportion of 1:1 (volume ratio), and fully and uniformly mixing. Incubate at 37℃for 2h.

4) And detecting by using 562nm wavelength on an enzyme label instrument, and calculating the protein concentration of the sample to be detected through a calibration curve.

The molecular weight of the obtained X fragment is 250kd, the molecular weight of the Y fragment is 150kd, the molecular weight of the D fragment is 100kd and the molecular weight of the E fragment is 50kd.

Specifically, the preparation of fibrinogen degradation fragment X, Y, D, E comprises the following steps:

1) Reagent preparation

Normal saline (0.9%): 0.9g of sodium chloride was weighed out and 100mL of purified water was added to dissolve completely.

Fibrinogen solution (1 mg/mL): 10mg of fibrinogen was weighed, and 10mL of physiological saline was added for complete dissolution. Wherein, the fibrinogen adopts commercial products, and the CAS number is 9001-32-5; the manufacturer: sigma; cargo number: F3879.

plasmin solution (150. Mu.g/ml): 150 μg of human plasmin was weighed and 1mL of physiological saline was added for complete dissolution. Wherein, the humanized plasmin adopts commercial products with CAS number 9001-90-5; the manufacturer: sigma; cargo number: p1867; enzyme activity: 2IU/mg.

Aprotinin solution (1 mg/mL): 10mg aprotinin was weighed and added to 10mL of physiological saline to dissolve thoroughly. Wherein, aprotinin is commercially available and has CAS number of 9087-70-1; the manufacturer: sigma; cargo number: a6103; enzyme activity: 5TIU/mg.

20% ethanol: 200ml of absolute ethyl alcohol is measured, 800ml of purified water is added, and the mixture is uniformly mixed.

Tris buffer (50 mM): 6.05g of tris (hydroxymethyl) aminomethane, 9g of sodium chloride and 800mL of purified water are weighed, the pH is adjusted to 8.4-8.6 by hydrochloric acid after the solution is completely dissolved, and the volume of the purified water is fixed to 1000mL.

SDS electrophoresis buffer: 14.4g glycine, 3g tris (hydroxymethyl) aminomethane, 1g SDS were weighed, dissolved in purified water and then fixed to 1000ml.

2) Degradation of fibrinogen

To 1mL of fibrinogen solution having a concentration of 1mg/mL, 20. Mu.L of plasmin solution having a concentration of 150. Mu.g/mL was added, and degradation was performed at 37℃for 30 minutes, and 10. Mu.L of aprotinin solution having a concentration of 1mg/mL was added to terminate the fibrinolysis reaction to obtain a mixed FDP.

3) Preparation of X fragment, Y fragment and D fragment

Performing SDS-PAGE on the mixed FDP obtained by degradation, performing electrophoresis by using 8% of separating gel and 5% of concentrating gel, performing electrophoresis by using 80V voltage for 5% of concentrating gel, performing electrophoresis by using 120V voltage for 8% of separating gel, performing dyeing for 30min after electrophoresis for 1h, and decolorizing to obtain a viewing result.

And respectively recovering the X fragment, the Y fragment and the D fragment by using a protein gel recovery kit, and carrying out protein concentration test by using a BCA protein concentration kit.

4) Preparation of E fragment

Purifying the degraded mixed FDP by using gel filtration chromatography, purifying by using superdex200 filler of GE molecular sieve, loading 5mL, and running 1 column volume with purified water at a flow rate of 1mL/min, balancing by running 1 column volume with Tris buffer solution, regulating ultraviolet UV to zero, observing conductivity rise, loading, collecting eluting sample, running 1 column volume with purified water, and preserving the purification column with 20% ethanol. Protein concentration testing was performed with BCA protein concentration kit.

As other embodiments of the application, the fibrinogen degradation fragment X, Y, D, E obtained by the preparation method can be used for preparing an FDP calibrator, wherein the fibrinogen degradation fragment X, Y, D, E is used as a calibrator; or subjecting plasma containing fibrinogen degradation products to FDP assay, adjusting to a concentration at which fibrinogen degradation fragment X, Y, D, E acts as a calibrator, including but not limited to dilution or concentration, based on the results; or preparing fibrinogen degradation fragment X, Y, D, E fragment into freeze-dried powder, and dissolving the fibrinogen degradation fragment X, Y, D, E fragment into a buffer solution for use.

Preferably, the calibration material prepared from the fibrinogen degradation fragment X, Y, D, E can comprise human plasma fibrinogen, a buffer solution, a protein stabilizer, a pH regulator, a preservative and the like. Wherein the buffer solution can be Tris buffer solution, glycine buffer solution, phosphate buffer solution and the like; the protein stabilizing agent may be bovine serum albumin; the pH adjustor can be a buffer solution such as phosphate buffer, borate buffer, boric acid buffer, etc.; the preservative can be benzoic acid and its salts, sorbic acid and its salts, dehydroacetic acid and sodium salts, nipagin esters, etc.

Example 1

The embodiment provides a preparation method of fibrinogen degradation fragment X, Y, D, E, which comprises the following steps:

1) Degradation of fibrinogen

2) Preparation of fragments X, Y and D

As shown in the SDS-PAGE result of FIG. 1, the X fragment 250kD, the Y fragment 150kD, the D fragment 100kD and the X fragment 50kD are respectively obtained, wherein the E fragment cannot be cleared and distinguished by the electrophoresis pattern due to the small molecular weight, and therefore, the separation is carried out by adopting a gel filtration chromatography method. The X fragment, Y fragment and D fragment were recovered respectively using a protein gel recovery kit, and protein concentration test was performed using a BCA protein concentration kit (Table 3).

TABLE 3 concentration of fragments X, Y, D (. Mu.g/ml)

4) Preparation of E fragment

Continuing to purify by using gel filtration chromatography, purifying by using superdex200 filler of GE molecular sieve, loading 5mL, running 1mL/min flow rate with purified water, running 1 column volume with Tris buffer solution for balancing, regulating ultraviolet UV to zero, observing conductivity rise, loading, collecting eluting sample, running 1 column volume with purified water, and preserving the purifying column with 20% ethanol.

As shown in FIG. 2 and FIG. 3, the molecular sieve of FIG. 2 shows that peak 1 is a large molecular fragment, peak 2 is a small molecular fragment, and the molecular sieve of FIG. 3 shows that peak 1 is a mixture of X, Y, D fragments, peak 2 is an E fragment and has a size of 50kD. Protein concentration tests were performed with BCA protein concentration kit and the results are shown in table 4.

TABLE 4 fragment E concentration (μg/ml)

The factors influencing the preparation of fibrinogen-degrading fragments according to the application are described below in connection with specific experiments.

Comparative example 1

The preparation method is the same as in example 1, except that: 1mL fibrinogen with the concentration of 0.5mg/mL is degraded, 10 mu L of plasmin solution with the concentration of 150 mu g/mL is added, degradation is carried out for 10min, and 10 mu L of aprotinin with the concentration of 1mg/mL is added.

As shown in FIG. 4, the molecular fragments degraded by measurement are about 245kd, the degradation time is short, the plasmin content and the fibrinogen concentration are low, and small molecular fragments such as Y fragments (150 kd), D fragments (100 kd) and E fragments (50 kd) are not degraded.

Example 2 time effect on FDP

The preparation method is the same as in example 1, except that: a fibrinogen solution (1 mL) with a concentration of 1mg/mL was added with 20. Mu.L of a plasmin solution (150. Mu.g/mL), and the mixture was degraded at 37℃to give a mixed FDP at 0, 10min, 20min, 30min, 40min, 60min and 90min, and the results were shown in Table 5, using a Fibrinogen Degradation Product (FDP) assay kit.

TABLE 5 FDP concentrations (. Mu.g/ml) obtained at different degradation times

As shown in Table 5, the degradation time of the present example was 30min, and the concentration of FDP obtained by degradation at 10min, 20min, 40min, and 90min was much less than 30min.

Example 3 effects of fibrinogen concentration on FDP

The preparation method is the same as in example 1, except that: 0.5mg/mL fibrinogen solution, 1mg/mL fibrinogen solution and 2mg/mL fibrinogen solution 1mL were prepared, 20. Mu.l of plasmin solution of 150. Mu.g/mL was added thereto, and the mixture was allowed to stand at 37℃for degradation for 30 minutes to obtain a mixed FDP, and the results were measured using a Fibrinogen Degradation Product (FDP) measuring kit, and are shown in Table 6.

TABLE 6 degradation of fibrinogen to FDP concentration (. Mu.g/ml) at various concentrations

As is clear from Table 6, the concentration of FDP obtained by the degradation was improved by 92.50% and 28.89% by adding 20. Mu.l of a plasmin solution having a concentration of 150. Mu.g/mL to 1mL of fibrinogen solution having a concentration of 1mg/mL, respectively, and by obtaining FDP having a concentration of optimum concentration by the degradation as compared with that obtained by 0.5mg/mL of fibrinogen solution and 2mg/mL of fibrinogen solution.

Example 4 Effect of plasmin amount on FDP

The preparation method is the same as in example 1, except that: to 1mL of a fibrinogen solution having a concentration of 1mg/mL, 0. Mu.l, 5. Mu.l, 10. Mu.l, 20. Mu.l, 30. Mu.l, 40. Mu.l of a plasmin solution having a concentration of 150 mg/mL were added, and the mixture was degraded at 37℃for 30 minutes to obtain a mixed FDP, and the result was measured using a Fibrinogen Degradation Product (FDP) measuring kit, and the results are shown in Table 7.

TABLE 7 degradation of fibrinogen to FDP concentration (. Mu.g/ml) with varying amounts of plasmin

As is clear from Table 7, when 20. Mu.l of a plasmin solution having a concentration of 150. Mu.g/mL was added to 1mL of a fibrinogen solution, FDP obtained by degradation had an optimal concentration, and the concentration of FDP obtained by degradation was directly affected by changing the amount of plasmin, FDP concentration was far less than 20. Mu.l when the amount of plasmin was 0. Mu.l, 5. Mu.l, 10. Mu.l, 30. Mu.l, 40. Mu.l.

As is clear from examples 2 to 4, the concentration of FDP obtained by degradation is far smaller than that of example 1 with the change of any one of degradation time, fibrinogen concentration and plasmin amount, and the reason for example 2 is that the degradation product is gradually degraded into other small molecule fragments with the extension of degradation time, which cannot be detected by FDP reagent, resulting in the decrease of FDP concentration. Examples 3 to 4 are because the degradation conditions are changed such that small molecule fragments such as Y fragment, D fragment, E fragment are not completely degraded, resulting in a decrease in FDP concentration.

Product performance detection

The X and Y fragments purified in example 1 were diluted to 10.+ -. 0.5. Mu.g/mL and 5.+ -. 0.5. Mu.g/mL with a buffer component and a lyoprotectant, respectively, to prepare FDP calibrator, which was tested for performance index such as reproducibility, inter-vial differences, and reconstitution stability using FDP detection reagents (antibody-recognizing X, Y fragments).

Table 8 repeatability

TABLE 9 difference between bottles

TABLE 10 reconstitution stability

As is clear from tables 8 to 10, the X and Y fragment repeatability CV obtained by the purification of the technical scheme of example 1 is within 5%, the inter-bottle difference CV is within 5%, and the reconstitution stability is 2-8 ℃ and the deviation is not more than + -10%.

Although embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A preparation method of fibrinogen degradation fragments is characterized in that the degradation fragments consist of an X fragment, a Y fragment, a D fragment and an E fragment, and the plasmin with the enzyme activity of 2IU per 1mg is adopted to prepare 20 mu l of plasmin solution with the concentration of 150 mu g/ml, and the plasmin solution is matched with 1ml of fibrinogen solution with the concentration of 1mg/ml for degradation reaction, and the degradation reaction is carried out for 30min at 37 ℃;

terminating the fibrinolysis degradation reaction process by aprotinin, wherein 1ml of fibrinogen solution is matched with 0.05TIU of aprotinin in the fibrinolysis reaction process;

separating and purifying the X fragment, the Y fragment and the D fragment by adopting polyacrylamide gel electrophoresis and a protein gel recovery method, wherein the polyacrylamide gel electrophoresis is that 8% of separation gel and 5% of concentration gel are adopted, the 8% of separation gel is electrophoresed under 120V voltage, and the 5% of concentration gel is electrophoresed under 80V voltage; the E fragment was isolated and purified by gel filtration chromatography.