CN117568445A - Preparation method and application of TAT (TAT) and PIC (PIC) composite quality control product - Google Patents
Preparation method and application of TAT (TAT) and PIC (PIC) composite quality control product Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/968—Plasmin, i.e. fibrinolysin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/974—Thrombin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a preparation method and application of a TAT and PIC composite quality control product, and relates to the technical field of biochemical detection reagents, animal thrombin is added into animal plasma, and thrombin and antithrombin III in the plasma are combined to form a thrombin-antithrombin composite; continuing to add glucokinase or urokinase or tissue plasminogen activator to the plasma to activate plasminogen in the plasma to form plasmin, and combining plasmin with alpha 2 plasmin in the plasma to form plasmin-alpha 2 plasmin inhibitor complex; after the reaction is completed, aprotinin is added to destroy the enzyme activity so as to terminate the enzymolysis reaction, and a TAT and PIC composite mother solution is obtained; then diluting the TAT and PIC composite mother liquor by using a quality control product diluent to obtain a composite quality control liquid; and finally, freeze-drying the composite quality control liquid to obtain the TAT and PIC composite quality control product. Provides a guarantee for the accuracy of the clinical test result, and solves the problems of large difference between test result batches and the like.
Description
Technical Field
The invention relates to the technical field of biochemical detection reagents, in particular to a preparation method and application of a TAT (TAT) and PIC composite quality control product.
Background
The quality control is a basis for ensuring the accuracy and reliability of the detection result, and the quality control product is an essential part of the quality control. Thrombotic diseases are the general names of various diseases such as pathological changes, necrosis, corresponding dysfunction and damage of corresponding organs and tissues caused by blood vessel blockage due to thrombosis, and can be classified into arterial thrombosis, venous thrombosis and micro-thrombosis according to the types of blood vessels generated by thrombosis, and have become the largest killers in cardiovascular and cerebrovascular diseases, and the pathological process mainly relates to three systems of vascular endothelium, blood coagulation and fibrinolysis.
Thrombin-antithrombin complex (TAT) is a complex formed by the 1:1 combination of thrombin and antithrombin iii, and thrombin is normally present in the blood in the form of a zymogen, and after activation of the coagulation system, prothrombin is converted to thrombin, so thrombin synthesis means the initiation of the coagulation system. Thrombin has a very short half-life in blood, only a few seconds, and is quickly neutralized by an anticoagulant substance AT-iii, so thrombin cannot be directly and accurately measured, instead thrombin-antithrombin complex TAT is detected, and thus TAT generation directly reflects activation of the coagulation system and serves as a marker for starting the coagulation system and a sensitivity index for anticoagulant therapy.
Plasmin-alpha 2 Plasmin Inhibitor Complex (PIC) is a complex formed by 1:1 combination of plasmin and alpha 2 plasmin inhibitor, and after activation of plasminogen by t-PA synthesized by endothelial cells, it is converted into plasmin, and the plasmin system is started up, and fibrin and fibrinogen are hydrolyzed, and its degradation products are FDP and D-dimer. At the same time plasmin is rapidly neutralized by the alpha 2 plasmin inhibitor (alpha 2 PI) in the blood, forming a plasmin-alpha 2 Plasmin Inhibitor Complex (PIC). The PIC directly reflects the generation of plasmin, overcomes the defects of short plasmin half-life and difficult detection, and is an index for activating plasmin and starting plasmin system.
The magnetic particle chemiluminescence technology is an enzyme immunity technology using a chemiluminescent agent as a substrate, and simultaneously uses nano-scale magnetic particles as a solid phase carrier, so that the adsorption area is increased, the antigen and the antibody are combined to the maximum extent, the combination reaction is performed under the condition of being similar to a liquid phase, and the magnetic particle chemiluminescence technology has the advantages of the chemiluminescence technology and the enzyme immunity technology, and is a non-radioactive detection method which is rapidly developed in recent years. The quality control product is used as an important component of the chemiluminescent reagent, and has the main function of monitoring the state of the whole detection system, confirming that the sample detection is carried out in the normal state of the detection system and providing a very important reference function for the detection result.
At present, quality control products for detecting thrombus diseases also have the problems of low accuracy of clinical test results and high resource consumption.
Disclosure of Invention
The invention aims to solve the technical problems that the quality control product for detecting the thrombus diseases has low accuracy of clinical test results and high resource consumption at present, and aims to provide a preparation method and application of a TAT and PIC composite quality control product, which solves the problems that the quality control product for detecting the thrombus diseases has low accuracy of clinical test results and high resource consumption.
The invention is realized by the following technical scheme:
in a first aspect, the invention provides a preparation method of a TAT and PIC composite quality control product, comprising the following steps:
step one, preparing TAT antigen: adding animal thrombin to the animal plasma, the thrombin combining with antithrombin III in the plasma to form a thrombin-antithrombin complex;
step two, preparing PIC antigen: continuing to add staphylokinase or urokinase or tissue plasminogen activator to the animal plasma to activate the plasmin in the plasma to form plasmin and combine the plasmin with alpha 2 plasmin in the plasma to form a plasmin-alpha 2 plasmin inhibitor complex;
Step three, finishing the enzymolysis reaction, and adding aprotinin to destroy the enzyme activity after the reaction is finished so as to terminate the enzymolysis reaction, thereby obtaining TAT and PIC composite mother liquor;
step four: preparing quality control product diluent;
step five: diluting the TAT and PIC composite mother liquor by using a quality control product diluent to obtain a TAT and PIC composite quality control liquid;
step six: and freeze-drying the composite quality control liquid to obtain the TAT and PIC composite quality control product.
The TAT and PIC composite quality control product prepared by the preparation method of the TAT and PIC composite quality control product has the advantages of good stability, low cost, convenient use, contribution to clinical test, no influence of transportation and temperature factors, good detection repeatability, capability of meeting the quality control requirements of clinical on TAT and PIC detection, capability of improving the accuracy of clinical sample detection results, provision of guarantee for the accuracy of clinical detection results, capability of greatly saving resources during clinical application, and capability of solving the problems of large inter-batch difference of the test results and the like.
The TAT and PIC composite quality control product prepared in the invention is an in vitro diagnostic reagent product, is a composite quality control product for detecting thrombus markers, namely thrombin-antithrombin III complex (TAT) and plasmin-alpha 2 Plasmin Inhibitor Complex (PIC), and is suitable for joint detection of thrombus indexes.
Wherein, in the first step, the temperature of mixing the plasma and the thrombin is 30-40 ℃, and in the second step, the plasmin in the plasma is activated at the temperature of 30-40 ℃ to form the plasmin. Of these, 37℃is preferred.
Further, the plasma in the first step is animal plasma, and thrombin and the plasma are derived from the same animal.
Further, the animal plasma includes porcine-derived plasma, bovine-derived plasma, or rabbit-derived plasma.
The plasma and the thrombin used in the invention are both derived from common animals, are easy to obtain and have low cost, and play a role in reducing the cost of producing the composite quality control product of thrombin-antithrombin III complex (TAT) and plasmin-alpha 2 Plasmin Inhibitor Complex (PIC).
Further, the addition amount of thrombin is 300U/mL-1000U/mL.
In the second step, the addition amount of the staphylokinase, urokinase or tissue plasminogen activator is 500U/mL-5000U/mL.
Further, in the fourth step, the components of the diluent include buffer solution, sodium chloride, serum albumin, mannitol and sodium azide.
Further, the buffer solution comprises a phosphate buffer solution or a tris buffer solution.
Further, the addition amount of the phosphate buffer solution was 20mM, and the addition amount of the tris buffer solution was 0.05mM.
Wherein the pH value of the buffer solution is controlled to 7.0-8.0. The pH is preferably 7.4.
Further, the addition amount of the sodium chloride is 0.9% (V/W); the addition amount of serum protein is 10-50 g/L; the addition amount of mannitol is 10-20 g/L, and the addition amount of sodium azide is 0.5g/L.
In a second aspect, the invention provides a detection kit, which comprises the TAT and PIC composite quality control product prepared by the preparation method.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The TAT and PIC composite quality control product prepared by the preparation method of the TAT and PIC composite quality control product has the advantages of good stability, low cost, convenient use, contribution to clinical test, no influence of transportation and temperature factors, good detection repeatability, capability of meeting the quality control requirements of clinical on TAT and PIC detection, capability of improving the accuracy of clinical sample detection results, provision of guarantee for the accuracy of clinical detection results, capability of greatly saving resources during clinical application, capability of solving the problems of large batch-to-batch difference of the test results and the like;
(2) The TAT and PIC composite quality control product prepared in the invention is an in-vitro diagnostic reagent product, is a composite quality control product for detecting thrombus markers, namely thrombin-antithrombin III complex (TAT) and plasmin-alpha 2 Plasmin Inhibitor Complex (PIC), and is suitable for joint detection of thrombus indexes;
(3) The plasma and the thrombin used in the invention are both derived from common animals, are easy to obtain and have low cost, and play a role in reducing the cost of producing the composite quality control product of thrombin-antithrombin III complex (TAT) and plasmin-alpha 2 Plasmin Inhibitor Complex (PIC).
Drawings
In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the drawings that are needed in the examples will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and that other related drawings may be obtained from these drawings without inventive effort for a person skilled in the art. In the drawings:
FIG. 1 is a process flow diagram of a method for preparing a TAT and PIC composite quality control product in embodiment 1 of the invention;
FIG. 2 is a graph showing the result of QC1 reconstitution stability verification in example 1 of the present invention;
FIG. 3 is a graph showing the result of QC2 reconstitution stability verification in example 1 of the present invention;
FIG. 4 is a graph showing the results of QC1 thermal acceleration stability verification in example 1 of the present invention;
FIG. 5 is a graph showing the results of QC2 thermal acceleration stability verification in example 1 of the present invention;
FIG. 6 is a graph showing the result of QC1 reconstitution stability verification in example 2 of the present invention;
FIG. 7 is a graph showing the results of QC2 reconstitution stability verification in example 2 of the present invention;
FIG. 8 is a graph showing the results of QC1 thermal acceleration stability verification in example 2 of the present invention;
FIG. 9 is a graph showing the results of QC2 thermal acceleration stability test in example 2 of the present invention;
FIG. 10 is a graph showing the results of QC1 reconstitution stability verification in example 3 of the present invention;
FIG. 11 is a graph showing the results of QC2 reconstitution stability verification in example 3 of the present invention;
FIG. 12 is a graph showing the results of QC1 thermal acceleration stability test in example 3 of the present invention;
FIG. 13 is a graph showing the results of QC2 thermal acceleration stability test in example 3 of the present invention;
FIG. 14 is a graph showing the results of QC1 reconstitution stability verification in example 4 of the present invention;
FIG. 15 is a graph showing the results of QC2 reconstitution stability verification in example 4 of the present invention;
FIG. 16 is a graph showing the results of QC1 thermal acceleration stability test in example 4 of the present invention;
FIG. 17 is a graph showing the results of QC2 thermal acceleration stability test in example 4 of the present invention;
FIG. 18 is a graph showing the results of QC1 reconstitution stability verification in example 5 of the present invention;
FIG. 19 is a graph showing the results of QC2 reconstitution stability verification in example 5 of the present invention;
FIG. 20 is a graph showing the results of QC1 thermal acceleration stability test in example 5 of the present invention;
FIG. 21 is a graph showing the results of QC2 thermal acceleration stability test in example 5 of the present invention;
FIG. 22 is a graph showing the results of QC1 reconstitution stability test in example 6 of the present invention;
FIG. 23 is a graph showing the results of QC2 reconstitution stability test in example 6 of the present invention;
FIG. 24 is a graph showing the results of QC1 thermal acceleration stability test in example 6 of the present invention;
FIG. 25 is a graph showing the results of QC2 thermal acceleration stability test in example 6 of the present invention;
FIG. 26 is a graph showing the results of QC1 reconstitution stability test in example 7 of the present invention;
FIG. 27 is a graph showing the results of QC2 reconstitution stability test in example 7 of the present invention;
FIG. 28 is a graph showing the results of QC1 thermal acceleration stability test in example 7 of the present invention;
FIG. 29 is a graph showing the results of QC2 thermal acceleration stability test in example 7 of the present invention;
FIG. 30 is a graph showing the results of QC1 reconstitution stability verification in example 8 of the present invention;
FIG. 31 is a graph showing the results of QC2 reconstitution stability test in example 8 of the present invention;
FIG. 32 is a graph showing the results of QC1 thermal acceleration stability test in example 8 of the present invention;
FIG. 33 is a graph showing the results of QC2 thermal acceleration stability test in example 8 of the present invention;
FIG. 34 is a graph showing the results of QC1 reconstitution stability test in example 9 of the present invention;
FIG. 35 is a graph showing the results of QC2 reconstitution stability test in example 9 of the present invention;
FIG. 36 is a graph showing the results of QC1 thermal acceleration stability test in example 9 of the present invention;
FIG. 37 is a graph showing the results of QC2 thermal acceleration stability test in example 9 of the present invention;
FIG. 38 is a graph showing the results of QC1 reconstitution stability test in example 10 of the present invention;
FIG. 39 is a graph showing the results of QC2 reconstitution stability test in example 10 of the present invention;
FIG. 40 is a graph showing the results of QC1 thermal acceleration stability test in example 10 of the present invention;
FIG. 41 is a graph showing the results of QC2 thermal acceleration stability test in example 10 of the present invention;
FIG. 42 is a graph showing the results of QC1 reconstitution stability test in example 11 of the present invention;
FIG. 43 is a graph showing the results of QC2 reconstitution stability test in example 11 of the present invention;
FIG. 44 is a graph showing the results of QC1 thermal acceleration stability test in example 11 of the present invention;
FIG. 45 is a graph showing the results of QC2 thermal acceleration stability test in example 11 of the present invention;
FIG. 46 is Table 1 in example 1 of the present invention;
FIG. 47 is Table 2 in example 1 of the present invention;
FIG. 48 is Table 3 in example 2 of the present invention;
FIG. 49 is Table 4 in example 3 of the present invention;
FIG. 50 is Table 5 in example 4 of the present invention;
FIG. 51 is Table 6 in example 5 of the present invention;
FIG. 52 is Table 7 in example 6 of the present invention;
FIG. 53 is Table 8 in example 7 of the present invention;
FIG. 54 is Table 9 in example 8 of the present invention;
FIG. 55 is Table 10 in example 9 of the present invention;
FIG. 56 is Table 11 in example 10 of the present invention;
FIG. 57 is Table 12 in example 11 of the present invention.
Description of the embodiments
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.
Example 1
As shown in fig. 1, this embodiment provides a preparation method of TAT and PIC composite quality control product, which comprises the following steps:
s1: preparing TAT and PIC composite antigen;
s1-1: preparing TAT antigen, adding 500U/mL swine thrombin into swine plasma, mixing the plasma with thrombin at 37 ℃, and combining the thrombin with antithrombin III in the plasma to form thrombin-antithrombin complex (TAT);
S1-2: preparing PIC antigen, adding 1000U/mL urokinase into pig animal plasma, activating plasmin in the plasma at 37 ℃ to form plasmin, and combining plasmin with alpha 2 plasmin inhibitor in pig animal plasma to form plasmin-alpha 2 Plasmin Inhibitor Complex (PIC);
s1-3: after the enzymolysis reaction is finished, 1000U/mL aprotinin is added to destroy the enzyme activity to terminate the enzymolysis reaction, a filter membrane with the diameter of 0.2 mu m is used for filtering the solution, and the obtained supernatant is TAT and PIC composite mother liquor.
S2: preparing TAT and PIC composite quality control products;
s2-1: preparing quality control product diluent, wherein the quality control product diluent comprises the following main components: 20mM sodium phosphate buffer pH 7.4, 0.9% (V/W) sodium chloride, 25g/L bovine serum albumin, 20g/L mannitol and 0.5g/L sodium azide (NaN) 3 ）；
S2-2: respectively diluting TAT and PIC composite mother liquor to target concentrations of quality control 1 (QC 1) and quality control 2 (QC 2) (shown in the following table 1) by using the quality control product diluent to obtain composite quality control liquid;
s2-3: split charging and freeze-drying are carried out on the prepared composite quality control liquid, wherein the freeze-drying procedure is as follows: primary drying (sublimation drying) and secondary drying (desorption drying), specific lyophilization parameters are: pre-freezing to-40deg.C for 1 hr; vacuum pumping to sublimate and dry for 4 hours; the temperature was raised to 25℃and desorption drying was carried out for 30 hours.
S2-4: and (5) capping and labeling the freeze-dried quality control product to obtain the TAT and PIC composite quality control product.
The performance of the TAT and PIC composite quality control prepared in example 1 above was examined.
1. Appearance and uniformity detection:
5 sets of TAT and PIC composite quality control products prepared by the method of the example 1 are randomly extracted and divided into A, B, C, D, E groups, 1mL of purified water is used for dissolution, and after inversion and uniform mixing, the mixture is stood for 10 minutes, and the liquid properties in each bottle are observed. The qualified product should be light yellow clear liquid without visible turbidity. The results of the verification are shown in table 2 below.
From the results in the table, it can be seen that the TAT and PIC composite quality control product prepared by the method in example 1 has a fast dissolution rate and high uniformity.
2. And (3) detecting the reconstitution stability:
10 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 1 are randomly extracted, wherein 5 sets of the TAT and PIC composite quality control products are dissolved again and then are placed in a refrigerator at 4 ℃,5 sets of the TAT and PIC composite quality control products are not dissolved in the refrigerator at 4 ℃, detection is carried out as a control after the TAT and PIC composite quality control products are dissolved again respectively at 7 days, 14 days, 21 days and 28 days (calculated by taking the signal value of the complex solute control products of the same day as 100%), and the signal retention rate is calculated, and the results are shown in figures 2 and 3.
As can be seen from fig. 2 and 3, the TAT and PIC composite quality control product prepared by the method of example 1 of the present invention has stable components, and the signal retention rate of the two components after being reconstituted for 28 days is still more than 95% compared with that of the reconstituted reagent on the same day.
3. Thermal acceleration stability detection:
randomly extracting 3 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 1, carrying out a 37 ℃ heat acceleration damage test, examining the same batch of quality control products stored at the temperature of 4 ℃ in a control group after the heat acceleration at the temperature of 37 ℃ is carried out for 1 day, 4 days and 7 days, and calculating the signal retention rate (calculated by taking the signal value of the control group as 100 percent), wherein the signal retention rate is shown in figures 4 and 5.
As can be seen from fig. 4 and 5, the TAT and PIC composite quality control product prepared by the method of example 1 of the present invention has stable components, and after 7 days of thermal acceleration at 37 ℃, the retention rate of the two components detection signals remains above 95%, which indicates that the TAT and PIC composite quality control product prepared by the method of the present invention has strong stability.
In conclusion, the detection results prove that the TAT and PIC composite quality control product prepared by the preparation method has the characteristics of high redissolution speed, high uniformity, high stability and the like, and the TAT and PIC components in the composite quality control product can exist stably for a long time, so that the TAT and PIC composite quality control product is suitable for joint detection of thrombus projects TAT and PIC indexes in a laboratory and is beneficial to efficient development of detection.
Example 2
Based on example 1, this example provides a preparation method of TAT and PIC composite quality control product, which is different from example 1 in that the pig-derived thrombin added in this example is 300U/mL, and other technical features are exactly the same as example 1.
The performance of the TAT and PIC composite quality control prepared in example 2 above was examined.
1. Appearance and uniformity detection:
5 sets of TAT and PIC composite quality control products prepared by the method of the example 2 are randomly extracted, are divided into A, B, C, D, E sets, are dissolved by using 1mL of purified water, are reversely and uniformly mixed, are stood for 10 minutes, and are observed for liquid properties in each bottle. The qualified product should be light yellow clear liquid without visible turbidity. The results of the verification are shown in table 3 below.
From the results in the table, it can be seen that the TAT and PIC composite quality control product prepared by the method in example 2 has a fast dissolution rate and high uniformity.
2. And (3) detecting the reconstitution stability:
10 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 2 are randomly extracted, wherein 5 sets of the TAT and PIC composite quality control products are dissolved again and then are placed in a refrigerator at 4 ℃,5 sets of the TAT and PIC composite quality control products are not dissolved in the refrigerator at 4 ℃, detection is carried out as a control after the TAT and PIC composite quality control products are dissolved again respectively at 7, 14, 21 and 28 days (calculated by taking the signal value of the complex solute control products of the same day as 100%), and the signal retention rate is calculated, and the results are shown in figures 6 and 7.
As can be seen from fig. 6 and 7, the TAT and PIC composite quality control product prepared by the method of example 2 of the present invention has stable components, and the signal retention rate of the two components after being reconstituted for 28 days is still more than 95% compared with that of the reconstituted reagent on the same day.
3. Thermal acceleration stability detection:
randomly extracting 3 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 2, performing a 37 ℃ heat acceleration damage test, observing the 37 ℃ heat acceleration for 1 day, 4 days and 7 days, and calculating the signal retention rate (calculated by taking the signal value of a control group as 100 percent), as shown in figures 8 and 9.
As can be seen from fig. 8 and 9, the TAT and PIC composite quality control product prepared by the method of example 2 of the present invention has stable components, and after 7 days of thermal acceleration at 37 ℃, the retention rate of the two-component detection signals remains above 95%, which indicates that the TAT and PIC composite quality control product prepared by the method of the present invention has strong stability.
In conclusion, the detection results prove that the TAT and PIC composite quality control product prepared by the preparation method has the characteristics of high redissolution speed, high uniformity, high stability and the like, and the TAT and PIC components in the composite quality control product can exist stably for a long time, so that the TAT and PIC composite quality control product is suitable for joint detection of thrombus projects TAT and PIC indexes in a laboratory and is beneficial to efficient development of detection.
Example 3
Based on example 1, this example provides a preparation method of TAT and PIC composite quality control product, which is different from example 1 in that the pig-derived thrombin added in this example is 1000U/mL, and other technical features are exactly the same as example 1.
The performance of the TAT and PIC composite quality control prepared in example 3 above was examined.
1. Appearance and uniformity detection:
5 sets of TAT and PIC composite quality control products prepared by the method of the example 3 are randomly extracted, are divided into A, B, C, D, E sets, are dissolved by using 1mL of purified water, are reversely and uniformly mixed, are stood for 10 minutes, and are observed for liquid properties in each bottle. The qualified product should be light yellow clear liquid without visible turbidity. The results of the verification are shown in table 4 below.
From the results in the table, it can be seen that the TAT and PIC composite quality control product prepared by the method in example 3 has a fast dissolution rate and high uniformity.
2. And (3) detecting the reconstitution stability:
10 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 3 are randomly extracted, wherein 5 sets of the TAT and PIC composite quality control products are dissolved again and then are placed in a refrigerator at 4 ℃,5 sets of the TAT and PIC composite quality control products are not dissolved in the refrigerator at 4 ℃, detection is carried out as a control after the TAT and PIC composite quality control products are dissolved again respectively at 7 days, 14 days, 21 days and 28 days (calculated by taking the signal value of the complex solute control products of the same day as 100%), and the signal retention rate is calculated, and the results are shown in figures 10 and 11.
As can be seen from fig. 10 and 11, the TAT and PIC composite quality control product prepared by the method of example 3 of the present invention has stable components, and the signal retention rate of the two components after being reconstituted for 28 days is still more than 95% compared with that of the reconstituted reagent on the same day.
3. Thermal acceleration stability detection:
randomly extracting 3 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 3, performing a 37 ℃ heat acceleration damage test, observing the 37 ℃ heat acceleration for 1 day, 4 days and 7 days, and calculating the signal retention rate (calculated by taking the signal value of a control group as 100 percent), as shown in figures 12 and 13.
From fig. 12 and 13, it can be seen that the TAT and PIC composite quality control product prepared by the method of example 3 of the present invention has stable components, and after 7 days of thermal acceleration at 37 ℃, the retention rate of the two component detection signals remains above 95%, which indicates that the TAT and PIC composite quality control product prepared by the method of the present invention has strong stability.
In conclusion, the detection results prove that the TAT and PIC composite quality control product prepared by the preparation method has the characteristics of high redissolution speed, high uniformity, high stability and the like, and the TAT and PIC components in the composite quality control product can exist stably for a long time, so that the TAT and PIC composite quality control product is suitable for joint detection of thrombus projects TAT and PIC indexes in a laboratory and is beneficial to efficient development of detection.
Example 4
Based on the embodiment 1, the embodiment provides a preparation method of a TAT and PIC composite quality control product, which is different from the embodiment 1 in that urokinase added in the embodiment is 500U/mL, and other technical characteristics are identical to those of the embodiment 1.
The performance of the TAT and PIC composite quality control prepared in example 4 above was examined.
1. Appearance and uniformity detection:
5 sets of TAT and PIC composite quality control products prepared by the method of example 4 are randomly extracted, are divided into A, B, C, D, E sets, are dissolved by using 1mL of purified water, are reversely and uniformly mixed, are stood for 10 minutes, and are observed for liquid properties in each bottle. The qualified product should be light yellow clear liquid without visible turbidity. The results of the verification are shown in table 5 below.
From the results in the table, it can be seen that the TAT and PIC composite quality control product prepared by the method in example 4 has a fast dissolution rate and high uniformity.
2. And (3) detecting the reconstitution stability:
10 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 4 are randomly extracted, wherein 5 sets of the TAT and PIC composite quality control products are dissolved again and then are placed in a refrigerator at 4 ℃,5 sets of the TAT and PIC composite quality control products are not dissolved in the refrigerator at 4 ℃, detection is carried out as a control after the TAT and PIC composite quality control products are dissolved again respectively at 7 days, 14 days, 21 days and 28 days (calculated by taking the signal value of the complex solute control products of the same day as 100%), and the signal retention rate is calculated, and the results are shown in figures 14 and 15.
As can be seen from fig. 14 and 15, the TAT and PIC composite quality control product prepared by the method of example 4 of the present invention has stable components, and the signal retention rate of the two components after being reconstituted for 28 days is still more than 95% compared with that of the reconstituted reagent on the same day.
3. Thermal acceleration stability detection:
randomly extracting 3 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 4, performing a 37 ℃ heat acceleration damage test, observing the 37 ℃ heat acceleration for 1 day, 4 days and 7 days, and calculating the signal retention rate (calculated by taking the signal value of a control group as 100 percent), as shown in figures 16 and 17.
From fig. 16 and 17, it can be seen that the TAT and PIC composite quality control product prepared by the method of example 4 of the present invention has stable components, and after 7 days of thermal acceleration at 37 ℃, the retention rate of the two component detection signals remains above 95%, which indicates that the TAT and PIC composite quality control product prepared by the method of the present invention has strong stability.
In conclusion, the detection results prove that the TAT and PIC composite quality control product prepared by the preparation method has the characteristics of high redissolution speed, high uniformity, high stability and the like, and the TAT and PIC components in the composite quality control product can exist stably for a long time, so that the TAT and PIC composite quality control product is suitable for joint detection of thrombus projects TAT and PIC indexes in a laboratory and is beneficial to efficient development of detection.
Example 5
Based on the embodiment 1, the embodiment provides a preparation method of a TAT and PIC composite quality control product, which is different from the embodiment 1 in that urokinase added in the embodiment is 5000U/mL, and other technical characteristics are identical to those of the embodiment 1.
The performance of the TAT and PIC composite quality control prepared in example 5 above was examined.
1. Appearance and uniformity detection:
randomly extracting 5 sets of TAT and PIC composite quality control products prepared by the method of the example 5, dividing the TAT and PIC composite quality control products into A, B, C, D, E sets, dissolving the TAT and PIC composite quality control products by using 1mL of purified water, mixing the TAT and PIC composite quality control products reversely, standing for 10 minutes after standing, and observing the liquid properties in each bottle. The qualified product should be light yellow clear liquid without visible turbidity. The results of the verification are shown in table 6 below.
From the results in the table, it can be seen that the TAT and PIC composite quality control product prepared by the method in example 5 has a fast dissolution rate and high uniformity.
2. And (3) detecting the reconstitution stability:
10 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 5 are randomly extracted, wherein 5 sets of the TAT and PIC composite quality control products are dissolved again and then are placed in a refrigerator at 4 ℃,5 sets of the TAT and PIC composite quality control products are not dissolved in the refrigerator at 4 ℃, detection is carried out as a control after the TAT and PIC composite quality control products are dissolved again respectively at 7, 14, 21 and 28 days (calculated by taking the signal value of the complex solute control products of the same day as 100%), and the signal retention rate is calculated, and the results are shown in figures 18 and 19.
As can be seen from fig. 18 and 19, the TAT and PIC composite quality control product prepared by the method of example 5 of the present invention has stable components, and the signal retention rate of the two components after being reconstituted for 28 days is still more than 95% compared with that of the reconstituted reagent on the same day.
3. Thermal acceleration stability detection:
randomly extracting 3 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 5, performing a 37 ℃ heat acceleration damage test, observing the 37 ℃ heat acceleration for 1 day, 4 days and 7 days, and calculating the signal retention rate (calculated by taking the signal value of a control group as 100 percent), as shown in figures 20 and 21.
As can be seen from fig. 20 and 21, the TAT and PIC composite quality control product prepared by the method of example 5 of the present invention has stable components, and after 7 days of thermal acceleration at 37 ℃, the retention rate of the two components detection signals remains above 95%, which indicates that the TAT and PIC composite quality control product prepared by the method of the present invention has strong stability.
In conclusion, the detection results prove that the TAT and PIC composite quality control product prepared by the preparation method has the characteristics of high redissolution speed, high uniformity, high stability and the like, and the TAT and PIC components in the composite quality control product can exist stably for a long time, so that the TAT and PIC composite quality control product is suitable for joint detection of thrombus projects TAT and PIC indexes in a laboratory and is beneficial to efficient development of detection.
Example 6
Based on the embodiment 1, the embodiment provides a preparation method of a TAT and PIC composite quality control product, which is different from the embodiment 1 in that staphylokinase is used to replace urokinase in the embodiment, and other technical characteristics are identical to those of the embodiment 1.
The performance of the TAT and PIC composite quality control prepared in example 6 above was examined.
1. Appearance and uniformity detection:
randomly extracting 5 sets of TAT and PIC composite quality control products prepared by the method of example 6, dividing the TAT and PIC composite quality control products into A, B, C, D, E sets, dissolving the TAT and PIC composite quality control products by using 1mL of purified water, mixing the TAT and PIC composite quality control products reversely, standing for 10 minutes after standing, and observing the liquid properties in each bottle. The qualified product should be light yellow clear liquid without visible turbidity. The results of the verification are shown in table 7 below.
From the results in the table, it can be seen that the TAT and PIC composite quality control product prepared by the method in example 6 has a fast dissolution rate and high uniformity.
2. And (3) detecting the reconstitution stability:
10 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 6 are randomly extracted, wherein 5 sets of the TAT and PIC composite quality control products are dissolved again and then are placed in a refrigerator at 4 ℃,5 sets of the TAT and PIC composite quality control products are not dissolved in the refrigerator at 4 ℃, detection is carried out as a control after the TAT and PIC composite quality control products are dissolved again respectively at 7, 14, 21 and 28 days (calculated by taking the signal value of the complex solute control products of the same day as 100%), and the signal retention rate is calculated, and the results are shown in figures 22 and 23.
As can be seen from fig. 22 and 23, the TAT and PIC composite quality control product prepared by the method of example 6 of the present invention has stable components, and the signal retention rate of the two components after being reconstituted for 28 days is still more than 95% compared with that of the reconstituted reagent on the same day.
3. Thermal acceleration stability detection:
randomly extracting 3 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 6, performing a 37 ℃ heat acceleration damage test, observing the 37 ℃ heat acceleration for 1 day, 4 days and 7 days, and calculating the signal retention rate (calculated by taking the signal value of a control group as 100 percent), as shown in figures 24 and 25.
As can be seen from fig. 24 and 25, the TAT and PIC composite quality control product prepared by the method of example 6 of the present invention has stable components, and after 7 days of thermal acceleration at 37 ℃, the retention rate of the two components detection signals remains above 95%, which indicates that the TAT and PIC composite quality control product prepared by the method of the present invention has strong stability.
In conclusion, the detection results prove that the TAT and PIC composite quality control product prepared by the preparation method has the characteristics of high redissolution speed, high uniformity, high stability and the like, and the TAT and PIC components in the composite quality control product can exist stably for a long time, so that the TAT and PIC composite quality control product is suitable for joint detection of thrombus projects TAT and PIC indexes in a laboratory and is beneficial to efficient development of detection.
Example 7
Based on example 1, this example provides a method for preparing a TAT/PIC composite quality control product, which differs from example 1 in that tissue plasminogen activator (tPA) is used instead of urokinase, and other technical features are exactly the same as in example 1.
The performance of the TAT and PIC composite quality control prepared in example 7 above was examined.
1. Appearance and uniformity detection:
5 sets of TAT and PIC composite quality control products prepared by the method of example 7 are randomly extracted, are divided into A, B, C, D, E sets, are dissolved by using 1mL of purified water, are reversely and uniformly mixed, are stood for 10 minutes, and are observed for liquid properties in each bottle. The qualified product should be light yellow clear liquid without visible turbidity. The results of the verification are shown in table 8 below.
From the results in the table, it can be seen that the TAT and PIC composite quality control product prepared by the method in example 7 has a fast dissolution rate and high uniformity.
2. And (3) detecting the reconstitution stability:
10 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 7 are randomly extracted, wherein 5 sets of the TAT and PIC composite quality control products are dissolved again and then are placed in a refrigerator at 4 ℃,5 sets of the TAT and PIC composite quality control products are not dissolved in the refrigerator at 4 ℃, detection is carried out as a control after the TAT and PIC composite quality control products are dissolved again respectively at 7, 14, 21 and 28 days (calculated by taking the signal value of the complex solute control products of the same day as 100%), and the signal retention rate is calculated, and the results are shown in figures 26 and 27.
As can be seen from fig. 26 and 27, the TAT and PIC composite quality control product prepared by the method of example 7 of the present invention has stable components, and the signal retention rate of the two components after being reconstituted for 28 days is still more than 95% compared with that of the reconstituted reagent on the same day.
3. Thermal acceleration stability detection:
randomly extracting 3 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 7, performing a 37 ℃ heat acceleration damage test, observing the 37 ℃ heat acceleration for 1 day, 4 days and 7 days, and calculating the signal retention rate (calculated by taking the signal value of the control group as 100 percent), as shown in figures 28 and 29.
As can be seen from fig. 28 and 29, the TAT and PIC composite quality control product prepared by the method of example 7 of the present invention has stable components, and after 7 days of thermal acceleration at 37 ℃, the retention rate of the two-component detection signals remains above 95%, which indicates that the TAT and PIC composite quality control product prepared by the method of the present invention has strong stability.
In conclusion, the detection results prove that the TAT and PIC composite quality control product prepared by the preparation method has the characteristics of high redissolution speed, high uniformity, high stability and the like, and the TAT and PIC components in the composite quality control product can exist stably for a long time, so that the TAT and PIC composite quality control product is suitable for joint detection of thrombus projects TAT and PIC indexes in a laboratory and is beneficial to efficient development of detection.
Example 8
Based on example 1, this example provides a preparation method of TAT and PIC composite quality control product, which is different from example 1 in that the amount of bovine serum albumin used in the main component of the quality control product diluent in this example is 10g/L, and other technical characteristics are exactly the same as example 1.
The performance of the TAT and PIC composite quality control prepared in example 8 above was examined.
1. Appearance and uniformity detection:
5 sets of TAT and PIC composite quality control products prepared by the method of the example 8 are randomly extracted, are divided into A, B, C, D, E sets, are dissolved by using 1mL of purified water, are reversely and uniformly mixed, are stood for 10 minutes, and are observed for liquid properties in each bottle. The qualified product should be light yellow clear liquid without visible turbidity. The results of the verification are shown in table 9 below.
From the results in the table, it can be seen that the TAT and PIC composite quality control product prepared by the method in example 8 has a fast dissolution rate and high uniformity.
2. And (3) detecting the reconstitution stability:
10 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 8 are randomly extracted, wherein 5 sets of the TAT and PIC composite quality control products are dissolved again and then are placed in a refrigerator at 4 ℃,5 sets of the TAT and PIC composite quality control products are not dissolved in the refrigerator at 4 ℃, detection is carried out as a control after the TAT and PIC composite quality control products are dissolved again respectively at 7 days, 14 days, 21 days and 28 days (calculated by taking the signal value of the complex solute control products of the same day as 100%), and the signal retention rate is calculated, and the results are shown in figures 30 and 31.
As can be seen from fig. 30 and 31, the TAT and PIC composite quality control product prepared by the method of example 8 of the present invention has stable components, and after the two components are redissolved for 28 days, the signal retention rate of the redissolved reagent is still 93% or more compared with that of the redissolved reagent of the same day, which is slightly lower than that of example 1.
3. Thermal acceleration stability detection:
randomly extracting 3 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 8, performing a 37 ℃ heat acceleration damage test, observing the 37 ℃ heat acceleration for 1 day, 4 days and 7 days, and calculating the signal retention rate (calculated by taking the signal value of a control group as 100 percent), as shown in figures 32 and 33.
As can be seen from fig. 32 and 33, the TAT and PIC composite quality control product prepared by the method of example 8 of the present invention has stable components, and after 7 days of thermal acceleration at 37 ℃, the retention rate of the two-component detection signals remains above 93%, which indicates that the TAT and PIC composite quality control product prepared by the method of the present invention has strong stability, but the thermal acceleration stability of this example is slightly worse than that of example 1.
In conclusion, the detection results prove that the TAT and PIC composite quality control product prepared by the preparation method has the characteristics of high redissolution speed, high uniformity, high stability and the like, and the TAT and PIC components in the composite quality control product can exist stably for a long time, so that the TAT and PIC composite quality control product is suitable for joint detection of thrombus projects TAT and PIC indexes in a laboratory and is beneficial to efficient development of detection.
Example 9
Based on example 1, this example provides a preparation method of TAT and PIC composite quality control product, which is different from example 1 in that the main component of the quality control product diluent of this example uses 50g/L bovine serum albumin, and other technical characteristics are exactly the same as example 1.
The performance of the TAT and PIC composite quality control prepared in example 9 above was examined.
1. Appearance and uniformity detection:
randomly extracting 5 sets of TAT and PIC composite quality control products prepared by the method of the example 9, dividing the TAT and PIC composite quality control products into A, B, C, D, E sets, dissolving the TAT and PIC composite quality control products by using 1mL of purified water, mixing the TAT and PIC composite quality control products reversely, standing for 10 minutes after standing, and observing the liquid properties in each bottle. The qualified product should be light yellow clear liquid without visible turbidity. The results of the verification are shown in table 10 below.
From the results in the table, it can be seen that the TAT and PIC composite quality control product prepared by the method in example 9 has a fast dissolution rate and high uniformity.
2. And (3) detecting the reconstitution stability:
10 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 9 are randomly extracted, wherein 5 sets of the TAT and PIC composite quality control products are dissolved again and then are placed in a refrigerator at 4 ℃,5 sets of the TAT and PIC composite quality control products are not dissolved in the refrigerator at 4 ℃, detection is carried out as a control after the TAT and PIC composite quality control products are dissolved again respectively at 7, 14, 21 and 28 days (calculated by taking the signal value of the complex solute control products of the same day as 100%), and the signal retention rate is calculated, and the results are shown in figures 34 and 35.
As can be seen from fig. 34 and 35, the TAT and PIC composite quality control product prepared by the method of example 9 of the present invention has stable components, and the signal retention rate of the two components after being reconstituted for 28 days is still more than 95% compared with that of the reconstituted reagent on the same day.
3. Thermal acceleration stability detection:
randomly extracting 3 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 9, performing a 37 ℃ heat acceleration damage test, observing the 37 ℃ heat acceleration for 1 day, 4 days and 7 days, and calculating the signal retention rate (calculated by taking the signal value of a control group as 100 percent), as shown in figures 36 and 37.
As can be seen from fig. 36 and 37, the TAT and PIC composite quality control product prepared by the method of example 9 of the present invention has stable components, and after 7 days of thermal acceleration at 37 ℃, the retention rate of the two components detection signals remains above 95%, which indicates that the TAT and PIC composite quality control product prepared by the method of the present invention has strong stability.
In conclusion, the detection results prove that the TAT and PIC composite quality control product prepared by the preparation method has the characteristics of high redissolution speed, high uniformity, high stability and the like, and the TAT and PIC components in the composite quality control product can exist stably for a long time, so that the TAT and PIC composite quality control product is suitable for joint detection of thrombus projects TAT and PIC indexes in a laboratory and is beneficial to efficient development of detection.
Example 10
Based on example 1, this example provides a preparation method of TAT and PIC composite quality control product, which is different from example 1 in that the main component of the quality control product diluent in this example uses 10g/L mannitol, and other technical characteristics are identical to those of example 1.
The performance of the TAT and PIC composite quality control prepared in example 10 above was examined.
1. Appearance and uniformity detection:
5 sets of TAT and PIC composite quality control products prepared by the method of example 10 are randomly extracted, are divided into A, B, C, D, E sets, are dissolved by using 1mL of purified water, are reversely and uniformly mixed, are stood for 10 minutes, and are observed for liquid properties in each bottle. The qualified product should be light yellow clear liquid without visible turbidity. The results of the verification are shown in table 11 below.
From the results in the table, it can be seen that the TAT and PIC composite quality control product prepared by the method in example 10 has a fast dissolution rate and high uniformity.
2. And (3) detecting the reconstitution stability:
10 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 10 are randomly extracted, wherein 5 sets of the TAT and PIC composite quality control products are dissolved again and then are placed in a refrigerator at 4 ℃,5 sets of the TAT and PIC composite quality control products are not dissolved in the refrigerator at 4 ℃, detection is carried out as a control after the TAT and PIC composite quality control products are dissolved again respectively at 7 days, 14 days, 21 days and 28 days (calculated by taking the signal value of the complex solute control products of the same day as 100%), and the signal retention rate is calculated, and the results are shown in figures 38 and 39.
As can be seen from fig. 38 and 39, the TAT and PIC composite quality control product prepared by the method of example 10 of the present invention has stable components, and after the two components are reconstituted for 28 days, the signal retention rate of the reconstituted reagent is still 93% or more compared with that of the reconstituted reagent of the same day, and the reconstituted stability is poorer than that of example 1.
3. Thermal acceleration stability detection:
randomly extracting 3 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 10, performing a 37 ℃ heat acceleration damage test, observing the 37 ℃ heat acceleration for 1 day, 4 days and 7 days, and calculating the signal retention rate (calculated by taking the signal value of a control group as 100 percent), as shown in figures 40 and 41.
As can be seen from fig. 40 and 41, the TAT and PIC composite quality control product prepared by the method of example 10 of the present invention has stable components, after 7 days of thermal acceleration at 37 ℃, the retention rate of the two-component detection signals remains above 93%, and the TAT and PIC composite quality control product prepared by the method of the present invention has stronger stability, but the thermal stability of this example is slightly worse than that of example 1.
In conclusion, the detection results prove that the TAT and PIC composite quality control product prepared by the preparation method has the characteristics of high redissolution speed, high uniformity, high stability and the like, and the TAT and PIC components in the composite quality control product can exist stably for a long time, so that the TAT and PIC composite quality control product is suitable for joint detection of thrombus projects TAT and PIC indexes in a laboratory and is beneficial to efficient development of detection.
Example 11
Based on example 1, this example provides a preparation method of TAT and PIC composite quality control product, which is different from example 1 in that the main component of the quality control product diluent in this example uses mannitol with an amount of 15g/L, and other technical characteristics are exactly the same as example 1.
The performance of the TAT and PIC composite quality control prepared in example 11 above was examined.
1. Appearance and uniformity detection:
5 sets of TAT and PIC composite quality control products prepared by the method of example 11 are randomly extracted, are divided into A, B, C, D, E sets, are dissolved by using 1mL of purified water, are reversely and uniformly mixed, are stood for 10 minutes, and are observed for liquid properties in each bottle. The qualified product should be light yellow clear liquid without visible turbidity. The results of the verification are shown in table 12 below.
From the results in the table above, it can be seen that the TAT and PIC composite quality control product prepared by the method in example 11 has a slower reconstitution rate than that in example 1.
2. And (3) detecting the reconstitution stability:
10 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 11 are randomly extracted, wherein 5 sets of the TAT and PIC composite quality control products are dissolved again and then are placed in a refrigerator at 4 ℃,5 sets of the TAT and PIC composite quality control products are not dissolved in the refrigerator at 4 ℃, detection is carried out as a control after the TAT and PIC composite quality control products are dissolved again respectively at 7 days, 14 days, 21 days and 28 days (calculated by taking the signal value of the complex solute control products of the same day as 100%), and the signal retention rate is calculated, and the results are shown in figures 42 and 43.
As can be seen from fig. 42 and 43, the TAT and PIC composite quality control product prepared by the method of example 11 of the present invention has stable components, and the signal retention rate of the two components after being reconstituted for 28 days is still more than 95% compared with that of the reconstituted reagent on the same day.
3. Thermal acceleration stability detection:
randomly extracting 3 sets of TAT and PIC composite quality control products prepared by the preparation method of the example 11, performing a 37 ℃ heat acceleration damage test, observing the 37 ℃ heat acceleration for 1 day, 4 days and 7 days, and calculating the signal retention rate (calculated by taking the signal value of the control group as 100 percent), as shown in figures 44 and 45.
From fig. 44 and 45, it can be seen that the TAT and PIC composite quality control product prepared by the method of example 11 of the present invention has stable components, and after 7 days of thermal acceleration at 37 ℃, the retention rate of the two component detection signals remains above 95%, which indicates that the TAT and PIC composite quality control product prepared by the method of the present invention has strong stability.
In conclusion, the detection results prove that the TAT and PIC composite quality control product prepared by the preparation method has the characteristics of high redissolution speed, high uniformity, high stability and the like, and the TAT and PIC components in the composite quality control product can exist stably for a long time, so that the TAT and PIC composite quality control product is suitable for joint detection of thrombus projects TAT and PIC indexes in a laboratory and is beneficial to efficient development of detection.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. The preparation method of the TAT and PIC composite quality control product is characterized by comprising the following steps of:
step one, preparing TAT antigen: adding animal thrombin to the animal plasma, the thrombin combining with antithrombin III in the plasma to form a thrombin-antithrombin complex;
step two, preparing PIC antigen: continuing to add staphylokinase or urokinase or tissue plasminogen activator to the animal plasma to activate the plasmin in the plasma to form plasmin and combine the plasmin with alpha 2 plasmin in the plasma to form a plasmin-alpha 2 plasmin inhibitor complex;
step three, finishing the enzymolysis reaction, and adding aprotinin to destroy the enzyme activity after the reaction is finished so as to terminate the enzymolysis reaction, thereby obtaining TAT and PIC composite mother liquor;
step four: preparing quality control product diluent;
step five: diluting the TAT and PIC composite mother liquor by using a quality control product diluent to obtain a TAT and PIC composite quality control liquid;
step six: and freeze-drying the composite quality control liquid to obtain the TAT and PIC composite quality control product.
2. The method for preparing TAT/PIC composite quality control according to claim 1, wherein the plasma in the first step is animal plasma, and thrombin and the plasma are derived from the same animal.
3. The method for preparing TAT/PIC composite quality control according to claim 2, wherein the animal plasma comprises porcine-derived plasma, bovine-derived plasma or rabbit-derived plasma.
4. The method for preparing the TAT/PIC composite quality control product according to claim 1, wherein the addition amount of thrombin is 300U/mL-1000U/mL.
5. The method for preparing a TAT/PIC composite quality control product according to claim 1, wherein in the second step, the addition amount of the staphylokinase, urokinase or tissue plasminogen activator is 500U/mL to 5000U/mL.
6. The method for preparing TAT and PIC composite quality control product according to claim 1, wherein in step four, the components of the diluent include buffer solution, sodium chloride, serum albumin, mannitol and sodium azide.
7. The method for preparing TAT and PIC composite quality control product according to claim 6, wherein the buffer solution comprises phosphate buffer solution or tris buffer solution.
8. The method for preparing the TAT/PIC composite quality control product according to claim 7, wherein the addition amount of the phosphate buffer solution is 20mM, and the addition amount of the tris buffer solution is 0.05mM.
9. The method for preparing the TAT/PIC composite quality control product according to claim 6, wherein the addition amount of sodium chloride is 0.9% (V/W); the addition amount of serum protein is 10-50 g/L; the addition amount of mannitol is 10-20 g/L, and the addition amount of sodium azide is 0.5g/L.
10. A detection kit, which is characterized by comprising the TAT and PIC composite quality control product prepared by the preparation method of any one of claims 1-9.
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