Fibrinogen quality control product and its preparation method and application

Abstract

The present invention relates to the field of biological detection, and in particular to a fibrinogen quality control product and a preparation method and application thereof. The raw materials of the fibrinogen quality control product of the present invention include blood, an anticoagulant, a PEG solution and a freeze-dried protective solution. The quality control product has good long-term stability after being reconstituted and freeze-dried. The experimental results show that the fibrinogen quality control product of the present invention is stable at room temperature after being reconstituted, and the commercial quality control product is stored at room temperature after opening the bottle and the concentration decreases from the seventh day, indicating that the fibrinogen quality control product of the present invention has better stability. Compared with the commercially available ones, the fibrinogen quality control product of the present invention has better stability after opening the bottle. For the clinical laboratory department of the hospital, especially the grassroots hospitals with a small business volume, in the quality control work of fibrinogen, the quality control product can be stored for a longer time after opening the bottle, which can reduce waste, reduce the detection cost, and bring convenience to the work. The freeze-dried product of the quality control product of the present invention is still relatively stable after 33 months, which is conducive to commercial production.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a fibrinogen quality control product and a preparation method and application thereof. Background Art

[0002] Coagulation: short for blood coagulation, is the process of blood changing from liquid state to gel state, and is an important part of the hemostatic function of mammals.

[0003] Fibrinogen is a central protein in the coagulation system. The final stage of coagulation is the conversion of fibrinogen into fibrin. Fibrinogen, also known as coagulation factor I, plays a key role in hemostasis and wound healing. It is a glycoprotein synthesized in the liver with an apparent molecular weight of 340,000 Da. It consists of two dimers, each of which is composed of three pairs of different disulfide-linked polypeptide chains, designated Aα, Bβ, and γ. Fibrinogen circulates in the bloodstream at concentrations of approximately 1.5 mg / ml to 4.0 mg / ml. When a blood vessel is damaged, platelets become activated and form a thrombus. Fibrinogen participates in the initial stages of hemostasis by promoting cross-linking of activated platelets.

[0004] The parallel activation of the coagulation chain reaction has been triggered. Fibrinogen is released as an end point by the proteolytic hydrolysis of fibrinopeptide A and fibrinopeptide B (at a slower rate) by thrombin, and is converted into fibrin. This soluble fibrin monomer is aggregated into double-stranded twisted fibrils. Subsequently, these fibrils are arranged in a transverse manner, making the fiber thicker. Then, these fibers are cross-linked to the fibrin network by FXIIIa, which interacts with the activated platelets and fibrin to form a stable platelet plug, thereby obtaining a stable clot.

[0005] Fibrin: During the coagulation process, thrombin cleaves fibrinopeptides A and B from fibrinogen to form monomeric proteins called fibrin monomers. Once formed, fibrin monomers begin to polymerize to form cross-linked fibrin. Simultaneously, insoluble fibrin is formed under the action of coagulation factor XIII. Fibrin monomers and insoluble fibrin are collectively referred to as fibrinogen. Existing fibrinogen quality control products on the market suffer from poor stability, are easily deteriorated, are expensive, and exhibit poor post-opening stability. Therefore, there is a need for new fibrinogen quality control products that are stable, resist deterioration, are affordable, and exhibit excellent post-opening stability. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a fibrinogen quality control product and its preparation method and application. The quality control product provided by the present invention has good stability, is not easy to deteriorate, is low in price, and has good stability after opening the bottle.

[0007] The present invention provides a fibrinogen quality control product, the raw materials of which include blood, anticoagulant, PEG solution and freeze-dried protective solution;

[0008] The freeze-drying protective solution includes water, B-400 aqueous defoamer, nicotinamide, HEPES, sucrose, sodium laurate, sodium perfluorooctanoate, thimerosal, and NaOH; wherein the mass ratio of the B-400 aqueous defoamer, nicotinamide, HEPES, sucrose, sodium laurate, sodium perfluorooctanoate, thimerosal, and NaOH is (5-15): (122.14-244.24): (119.2-238.3): (100-200): (0.55-0.111): (0.11-0.218): (2.5-5.0). The pH is adjusted to 7.3-7.6 with NaOH.

[0009] In some embodiments, in the fibrinogen quality control product described in the present invention, the lyophilized protective solution includes 5g / L~15g / L B-400 aqueous defoamer, 122.14g / L~244.24g / L nicotinamide, 119.2g / L~238.3g / L HEPES, 100g / L~200g / L sucrose, 0.55g / L~0.111g / L sodium laurate, 0.1100g / L~0.2180g / L sodium perfluorooctanoate, 2.5g / L~5.0g / L thimerosal, and the pH value is adjusted to 7.40 with 10M NaOH.

[0010] In other embodiments, the concentration of the lyophilization protective solution is 10 g / L B-400 aqueous defoamer, 244.24 g / L nicotinamide, 238.3 g / L HEPES, 200 g / L sucrose, 0.111 g / L sodium laurate, 0.2180 g / L sodium perfluorooctanoate, 5.0 g / L thimerosal and 10 M NaOH, and the pH value is adjusted to 7.40 with 10 M NaOH.

[0011] In the fibrinogen quality control product described in the present invention, the blood is pig blood.

[0012] In the fibrinogen quality control product of the present invention, the pH value of the quality control product is 7.3 to 7.6. In some embodiments, the pH value of the quality control product is 7.4.

[0013] In the fibrinogen quality control product described herein, the anticoagulant is a 2 wt% to 5 wt% aqueous solution of trisodium citrate. In some embodiments, the anticoagulant is 3.8 wt% trisodium citrate. The blood containing the anticoagulant is centrifuged for 30 minutes at a relative centrifugal force of 2000 g, and the upper plasma layer is carefully separated and aspirated.

[0014] In the fibrinogen quality control product described herein, the PEG solution is a 20 wt% to 50 wt% aqueous solution of PEG 4000. In some embodiments, a 30 wt% PEG 4000 solution, 6 wt% by volume of porcine plasma, is added to precipitate fibrinogen. Plasma is then added to dissolve the precipitate.

[0015] In the fibrinogen quality control product of the present invention, the amount of the PEG solution added is 4% to 12% of the blood volume; the volume ratio of the blood containing the anticoagulant to the freeze-dried protective solution is (0.75 to 0.8): (0.2 to 0.25).

[0016] The present invention provides a fibrinogen quality control product, the raw materials of which include blood, an anticoagulant, a PEG solution and a freeze-dried protective solution; the advantages of the fibrinogen quality control product of the present invention are good stability after reconstitution, good long-term stability of the freeze-dried product, and the freeze-dried product is still relatively stable after 33 months, which is conducive to commercial production. Experiments have found that during the preparation of the quality control product, the selection of anticoagulants, PEG and freeze-dried protective solution has a significant impact on the quality of the quality control product. The applicant optimizes the selection and dosage of each component, and ultimately makes the selection of each component reasonable, the concentration appropriate, and the components support, cooperate and interact with each other to obtain good stability. The present invention also provides a method for preparing the fibrinogen quality control product, which includes mixing blood containing an anticoagulant with a PEG solution, precipitating fibrinogen, adding blood containing an anticoagulant and the freeze-dried protective solution, and freeze-drying to obtain a fibrinogen quality control product.

[0017] In the method of the present invention, the method for preparing the blood containing the anticoagulant includes: mixing the blood with the anticoagulant and then centrifuging to obtain plasma; the volume ratio of the blood to the anticoagulant is 9:1.

[0018] In the method of the present invention, the centrifugal conditions include 2000g to 2500g and the centrifugal time is 20min to 40min.

[0019] The fibrinogen quality control product described in the present invention comprises raw materials including blood, anticoagulant, PEG solution and freeze-dried protective solution. The quality control product is stable after redissolution, and the freeze-dried product has good long-term stability. Experimental results show that the fibrinogen quality control product prepared by the present invention is relatively stable under room temperature storage conditions after redissolution, while the commercially available quality control product begins to decrease in concentration from the seventh day after opening the bottle for storage at room temperature, indicating that the fibrinogen quality control product prepared by the invention has better stability. Compared with commercially available products, the fibrinogen quality control product prepared by the present invention has better stability after opening the bottle. For clinical laboratories in hospitals, especially grassroots hospitals with smaller business volumes, in the quality control work of fibrinogen, the quality control product can be stored for a longer time after opening the bottle, which can reduce reagent waste, reduce detection costs, and bring convenience to the work. The freeze-dried fibrinogen quality control product prepared by the invention is still relatively stable after 33 months, which is conducive to commercial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive work:

[0021] Figure 1 The results of the stability test of the fibrinogen quality control product of the present invention and the commercial fibrinogen quality control product after opening the bottle are shown;

[0022] Figure 2 The long-term stability test results of the fibrinogen quality control product of the present invention and the commercial product are shown. DETAILED DESCRIPTION

[0023] The present invention provides a fibrinogen quality control product and its preparation method and application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0024] The purpose of the present invention is to provide a method for preparing a fibrinogen quality control product by using porcine plasma.

[0025] To achieve the above object, the present invention adopts the following technical solutions:

[0026] Porcine plasma is placed in a plastic beaker and PEG solution is added to precipitate fibrinogen. Plasma is then added to dissolve the precipitate. The PEG-fibrinogen precipitate is diluted with plasma containing lyophilization solution and then freeze-dried to produce a long-term stable fibrinogen quality control.

[0027] The plasma described herein is porcine plasma, which is blood collected from the jugular vein of a pig. An anticoagulant, typically 2-5% trisodium citrate, is added to the blood at a ratio of 9:1. The anticoagulant is centrifuged for 30 minutes at a relative centrifugal force of 2000-2500 g, and the upper plasma layer is carefully separated and aspirated.

[0028] The added PEG solution is 20% to 50% PEG4000 solution, and the added amount is 4% to 12% of the original plasma volume.

[0029] The formula of the freeze-dried protective solution is that the volume ratio of the freeze-dried protective solution to the plasma is (0.2-0.25):(0.75-0.8).

[0030]

[0031] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0032] Example 1

[0033] 1. Blood was collected from the jugular vein of a pig and an anticoagulant (3.8 wt% sodium citrate trihydrate solution) was added at a ratio of 9:1. The blood was centrifuged for 30 minutes at a relative centrifugal force of 2000 g, and the upper plasma layer was carefully separated and aspirated.

[0034] 2. Place a certain amount of the upper layer of porcine plasma in a plastic beaker and add 6% of the porcine plasma volume in a 30% PEG4000 solution to precipitate fibrinogen. Then, add plasma containing lyophilization protection solution (where the volume fraction of lyophilization protection solution is 25%) to dissolve the precipitate. After lyophilization, obtain a quality control sample.

[0035] The formula of the freeze-dried protective solution is as follows:

[0036]

[0037] Comparative Example 1

[0038] Steps 1 and 2 are the same as in Example 1, and the freeze-drying procedure is the same as in Example 1. The formula of the freeze-drying protective solution is as follows:

[0039]

[0040] Effect verification

[0041] The stability of the fibrinogen quality control product was tested using Example 1, Comparative Example 1 and a commercially available quality control product as samples.

[0042] Open bottle stability test: The fibrinogen quality control product prepared in Example 1 was accurately re-dissolved and stored at room temperature (20°C to 28°C). Samples were taken every 24 hours to test the fibrinogen quality control product concentration. Figure 1 , Table 1. The results show that the fibrinogen quality control product prepared by the present invention is relatively stable when stored at room temperature after reconstitution, while the concentration of the commercial quality control product decreases from the seventh day after opening the bottle and storing at room temperature, indicating that the fibrinogen quality control product prepared by the present invention has better stability.

[0043] Table 1: Open bottle stability test results

[0044]

[0045] The regression equation of the quality control product of the present invention is: y=0.484x+803.5, and a two-sided t test is performed on the significant difference between the slope b and 0. According to statistical calculation, t b =1.01, P>0.05, indicating that the fibrinogen quality control prepared by the present invention is basically stable after reconstitution and storage at room temperature for 10 days. The regression equation of the commercial quality control product is: y=2.018x+810.0t b =7.09, P < 0.05, indicating that the fibrinogen concentration of the commercially available quality control product changed significantly after being opened and stored at room temperature for 10 days. Figure 1 It can be seen that the fibrinogen concentration decreased significantly from day 7. The regression equation of the control product is y = -5.182x + 798.82t b =13.54, P < 0.05, indicating that the stability of the quality control product after removing some protective agents was poor.

[0046] It can be seen that the fibrinogen quality control product prepared by the present invention has better stability after opening the bottle than the commercially available product. For the clinical laboratory department of the hospital, especially the grassroots hospitals with smaller business volume, in the quality control work of fibrinogen, the quality control product can be stored for a longer time after opening the bottle, which can reduce reagent waste, reduce testing costs, and bring convenience to the work.

[0047] 2. Long-term stability test: The lyophilized fibrinogen quality control product prepared in Example 1 was stored at 2-8°C. One bottle was taken out every 3 months for accurate reconstitution and the concentration of the fibrinogen quality control product was tested. Figure 2 , Table 2. The results show that the lyophilized fibrinogen quality control product prepared by the present invention is still relatively stable after 33 months, which is conducive to commercial production.

[0048] Table 2: Long-term qualitative test results

[0049]

[0050] By linear regression analysis, y = 0.0407x + 805.85, a two-sided t test was performed on the significant difference between the slope b and 0. According to statistical calculations, t b =0.3918, P>0.05, indicating that the fibrinogen quality control product prepared by the present invention is basically stable when stored at +2 to +8°C for 33 months, has good long-term stability, and is conducive to commercial production.

[0051] The regression equation of commercially available quality control products is: y = -0.842x + 816.16t b =5.572, P < 0.05, indicating that the fibrinogen concentration of the commercially available quality control product changed significantly after being stored at +2 to +8°C for 33 months, and the Figure 2 It can be seen that from the 27th month, the fibrinogen concentration began to decrease significantly.

[0052] The regression equation for the control product is y = -1.536x + 817.99t b =9.544, P<0.05, indicating that the stability of the quality control product after removing some protective agents is poor.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fibrinogen quality control product, characterized in that: Its raw materials are blood, anticoagulant, PEG solution and freeze-dried protective solution; The blood is pig blood; The anticoagulant is a 2wt% to 5wt% trisodium citrate aqueous solution; The PEG solution is a 20wt% to 50wt% PEG4000 aqueous solution; The freeze-drying protective solution includes water, B-400 aqueous defoamer, nicotinamide, HEPES, sucrose, sodium laurate, sodium perfluorooctanoate, thimerosal and NaOH; wherein the mass ratio of the B-400 aqueous defoamer, nicotinamide, HEPES, sucrose, sodium laurate, sodium perfluorooctanoate and thimerosal is (5-15): (122.14-244.24): (119.2-238.3): (100-200): (0.55-0.111): (0.11-0.218): (2.5-5.0), and the pH is adjusted to 7.3-7.6 with NaOH.

2. The fibrinogen quality control product according to claim 1, characterized in that: The freeze-drying protective solution includes 5g / L to 15g / L B-400 aqueous defoamer, 122.14g / L to 244.24g / L nicotinamide, 119.2g / L to 238.3g / L HEPES, 100g / L to 200g / L sucrose, 0.55g / L to 0.111g / L sodium laurate, 0.1100g / L to 0.2180g / L sodium perfluorooctanoate, and 2.5g / L to 5.0g / L thimerosal, and the pH value is adjusted to 7.40 with NaOH.

3. The fibrinogen quality control product according to claim 1, characterized in that: The amount of PEG solution added is 4% to 12% of the blood volume; The volume ratio of the blood containing the anticoagulant to the freeze-dried protective solution is (0.75-0.8):(0.2-0.25).

4. The method for preparing the fibrinogen quality control product according to claim 1, characterized in that: The method comprises mixing blood containing an anticoagulant with a PEG solution, precipitating fibrinogen, adding blood containing an anticoagulant and the freeze-dried protective solution, and freeze-drying to obtain a fibrinogen quality control product.

5. The method according to claim 4, characterized in that The method for preparing the blood containing the anticoagulant comprises: mixing the blood with the anticoagulant and then centrifuging to obtain plasma; the volume ratio of the blood to the anticoagulant is 9:

1.

6. The method according to claim 5, characterized in that The centrifugal conditions include 2000g to 2500g and a centrifugal time of 20min to 40min.

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