Prothrombin time detection or determination kit as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medical reagents, and provides a prothrombin time detection or determination kit as well as a preparation method and application thereof, and the kit comprises the following components in percentage by mass: 3-5% of recombinant tissue factor ester, 0.7-1.2% of a stabilizer, 0.08-0.15% of a preservative and the balance of a solvent. The kit has good precision and stability, can be used for replacing a kit with rabbit brain powder or rabbit brain tissue fluid as a main component, and expands the variety of kits.

Description

Technical Field

[0001] The present application belongs to the technical field of medical reagents, and relates to a prothrombin time detection or determination kit and a preparation method and application thereof. Background Art

[0002] The prothrombin time detection or determination kit is used to determine the time required for plasma coagulation. The full English name of the prothrombin time detection or determination kit is ThrombopLastin-DS, which is abbreviated as PT in English.

[0003] Prothrombin time (PT) measurement is a screening test to check whether there is any dysfunction in the body's exogenous coagulation system function, and is also an important monitoring indicator for clinical anticoagulant treatment.

[0004] The detection or determination principle of the current prothrombin time detection or determination kit is to add excessive tissue thromboplastin and calcium ions to the plasma to be tested, so that prothrombin is converted into thrombin, and thrombin converts fibrinogen into fibrin clots, and the time required for coagulation is measured, which is the prothrombin time (PT) of the plasma to be tested. PT is negatively correlated with the content of exogenous coagulation factors in plasma.

[0005] The main components of the prothrombin time detection or determination kits currently used are basically rabbit brain powder or rabbit brain tissue fluid. For example, Formula 1 is: 0.3g / L trisodium citrate, 4.2g / L sodium chloride, 1.4g / L calcium gluconate, 3g / L bovine serum albumin, 25g / L mannitol, 20g / L rabbit brain powder; Formula 2 is: rabbit brain tissue fluid, calcium ions; Formula 3 is: thromboplastin: 5*8.5ml rabbit brain thromboplastin, containing stabilizer, polybrene and buffer. Buffer: 5*8.5ml buffer, containing calcium chloride and preservatives. Formula 4 is: containing rabbit brain extract, buffer, stabilizer and calcium chloride; Formula 5 is: 2% rabbit brain powder, 0.01% calcium chloride, 0.625% sodium chloride, 2% bovine serum albumin. It can be seen from this that the raw materials of existing prothrombin time detection or determination kits are too single, mainly relying on rabbit brain powder or rabbit brain tissue fluid, and have the problem of poor substitutability. Summary of the invention

[0006] The purpose of the present application is to provide a prothrombin time detection or determination kit and its preparation method and application, aiming to solve the problem that the main components of the current prothrombin time detection or determination kit are too single and have poor substitutability.

[0007] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0008] In a first aspect, the present application provides a prothrombin time detection or determination kit, comprising the following components in percentage by mass: 3-5% recombinant tissue factor ester, 0.7-1.2% stabilizer, 0.08-0.15% preservative, and the remainder being solvent.

[0009] In a second aspect, the present application provides a method for preparing the prothrombin time detection or determination kit, wherein a stabilizer and a preservative are dispersed in a solvent, and then recombinant tissue factor ester is added thereto and stirred evenly.

[0010] The beneficial effects of the present invention are:

[0011] Provided is a prothrombin time detection or determination kit mainly composed of recombinant tissue factor esters. The kit has good precision and stability and can be used to replace kits whose main components are rabbit brain powder or rabbit brain tissue fluid, thereby expanding the types of kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0013] Figure 1 is a correlation diagram of the lyophilized reagent in the embodiment of the present application and the comparative example 1;

[0014] Figure 2 It is a correlation diagram of the liquid reagent in the embodiment of the present application and the comparative example 1. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0016] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0017] The raw materials of existing prothrombin time detection or determination kits are too single, mainly relying on rabbit brain powder or rabbit brain tissue fluid, and have the problem of poor substitutability.

[0018] In view of the above problems, the present application provides a prothrombin time detection or determination kit, which mainly includes recombinant tissue factor ester.

[0019] Recombinant tissue factor ester refers to an ester obtained by esterifying recombinant human tissue factor, wherein the recombinant human tissue factor is generally an activated tissue factor prepared by esterifying a recombinant protein (rhTF243) with an extracellular domain and a transmembrane domain that is highly active, stable, and structurally uniform and that is efficiently expressed by Escherichia coli and a synthetic phospholipid.

[0020] The detection principle of the prothrombin time test or determination kit is: adding excess tissue thromboplastin and calcium ions (such as calcium chloride) to platelet-deficient plasma to convert prothrombin into thrombin, and thrombin converts fibrinogen into fibrin clots, resulting in the time required for plasma coagulation, which is the prothrombin time (PT) of the plasma sample to be tested. PT is negatively correlated with the content of exogenous coagulation factors in plasma.

[0021] Therefore, recombinant tissue factor ester can completely replace rabbit brain powder or rabbit brain tissue fluid to obtain new test kits, expanding the types of test kits and their risk resistance.

[0022] Generally, the mass percentage of the recombinant tissue factor ester in the kit is 3-5%, preferably 4-5%, and more preferably 5%.

[0023] In the present invention, the kit may also contain a stabilizer for maintaining the structural stability of the recombinant tissue factor ester. Generally, the stabilizer may be an animal serum albumin commonly used in the art, such as bovine serum albumin, human serum albumin, and the like.

[0024] Generally, the mass percentage of the stabilizer in the kit is 0.7-1.2%, preferably 0.9-1.1%, and more preferably 1.1%.

[0025] In the present invention, in order to further extend the shelf life of the kit, a preservative can also be added to the kit. In general, the preservative can be a common and commonly used reagent in the art, for example: Pc-300, Pc-300 is a ProCide series liquid preservative, which is one of the most widely used bactericides in the IVD (English full name is In Vitro Diagnosis) industry, and has the characteristics of safe use, environmental protection, and stability. It can effectively inhibit the growth of bacteria and fungi, and help extend the shelf life of IVD reagents by killing microorganisms with a broad spectrum. The ProCide series of liquid preservatives can efficiently penetrate the microbial cell membrane, inhibit cell respiration-related enzymes, and then quickly inhibit the activity of microbial cells. ProCide preservatives are safe and environmentally friendly at the recommended usage level, and the product reduces health hazards, toxicology or disposal problems. At the same time, its efficacy is better than that of traditional preservatives such as thimerosal and sodium azide.

[0026] Generally, the mass percentage of the preservative in the kit is 0.08-0.15%, preferably 0.09-0.12%, and more preferably 0.10%.

[0027] It should be noted that, in addition to the above components, the prothrombin time detection or determination kit disclosed in the present invention also includes a solvent. The common solvent in the art is: pure water, and the "pure water" here refers to deionized water.

[0028] In the present invention, the aforementioned kit may further include glycine, and the mass percentage of the glycine is generally 7-9%, preferably 8-9%, and more preferably 9%.

[0029] In the present invention, the aforementioned kit also includes anhydrous calcium chloride, and the anhydrous calcium chloride and the recombinant tissue factor ester participate in the coagulation reaction together and are used for the determination of prothrombin time.

[0030] Generally, the mass percentage of anhydrous calcium chloride is 1.5-2.0%, preferably 1.8-2.0%, and more preferably 2.0%.

[0031] It should be noted that the dosage form of the prothrombin time detection or determination kit disclosed in the present invention can be a lyophilized dosage form or a liquid dosage form. It should be noted that the lyophilized form needs to be configured with a corresponding resurrection liquid, which can be a resurrection liquid commonly used in the art and will not be described in detail here.

[0032] As a preferred embodiment, a prothrombin time detection or determination kit, a 1L volume kit comprises the following components: 9g glycine, 1.8g anhydrous calcium chloride, 0.8g bovine serum albumin, 1mL Pc-300, 40ml tissue factor ester, and the balance is pure water.

[0033] In order to obtain a kit with a long shelf life and stable performance, the present invention also provides a method for preparing the aforementioned prothrombin time detection or determination kit, comprising the following steps: dispersing a stabilizer and a preservative in a solvent, and then adding recombinant tissue factor ester thereto and stirring evenly.

[0034] When the kit contains glycine and / or anhydrous calcium chloride, the steps for preparing the prothrombin time detection or determination kit are: dispersing the stabilizer and preservative in a solvent, then adding glycine thereto for dispersion, and finally stirring the anhydrous calcium chloride and recombinant tissue factor ester evenly.

[0035] When preparing a lyophilized dosage form kit, in addition to the above steps, it also includes a lyophilization step and preparation of a resurrection solution. The equipment and operating parameters involved in the lyophilization step are conventional choices in the field and are not described in detail here.

[0036] It should be noted that the freeze-dried test kit and the resurrection solution are stored separately and are mixed together when needed.

[0037] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0038] Example 1

[0039] This embodiment discloses a prothrombin time detection or determination kit. The composition of the kit with a volume of 1L is as follows: 9g of glycine, 1.8g of anhydrous calcium chloride, 0.8g of bovine serum albumin, 1mL of Pc-300, 40ml of tissue factor ester, and pure water.

[0040] Among them, tissue factor ester is an activated tissue factor made by esterification of recombinant protein (rhTF243) and synthetic phospholipids.

[0041] This embodiment also discloses a method for preparing a prothrombin time detection or determination kit, comprising the following steps: dispersing bovine serum albumin and Pc-300 in pure water, then adding glycine into the system for dispersion, and finally adding anhydrous calcium chloride and recombinant tissue factor ester and stirring evenly.

[0042] Then, liquid type kit and freeze-dried type kit were prepared respectively.

[0043] Example 2

[0044] This embodiment discloses a prothrombin time detection or determination kit, which is composed of: 1.8 g of anhydrous calcium chloride, 0.8 g of bovine serum albumin, 1 mL of Pc-300, 40 ml of tissue factor ester, and pure water.

[0045] Among them, tissue factor ester is an activated tissue factor made by esterification of recombinant protein (rhTF243) and synthetic phospholipids.

[0046] This embodiment also discloses a method for preparing a prothrombin time detection or determination kit, comprising the following steps: dispersing bovine serum albumin and Pc-300 in pure water, and then adding anhydrous calcium chloride and recombinant tissue factor ester and stirring evenly.

[0047] Example 3

[0048] This embodiment discloses a prothrombin time detection or determination kit, which is composed of: 1.8 g of anhydrous calcium chloride, 40 ml of tissue factor ester, and pure water.

[0049] Among them, tissue factor ester is an activated tissue factor made by esterification of recombinant protein (rhTF243) and synthetic phospholipids.

[0050] This embodiment also discloses a method for preparing a prothrombin time detection or determination kit, comprising the following steps: adding anhydrous calcium chloride and recombinant tissue factor ester and stirring them evenly.

[0051] Example 4

[0052] This embodiment discloses a prothrombin time detection or determination kit, which is composed of: 9 g of glycine, 1.8 g of anhydrous calcium chloride, 0.8 g of human serum albumin, 1 mL of Pc-300, 40 ml of tissue factor ester, and pure water.

[0053] Among them, tissue factor ester is an activated tissue factor made by esterification of recombinant protein (rhTF243) and synthetic phospholipids.

[0054] This embodiment also discloses a method for preparing a prothrombin time detection or determination kit, comprising the following steps: dispersing human serum albumin and Pc-300 in pure water, then adding glycine into the system for dispersion, and finally adding anhydrous calcium chloride and recombinant tissue factor ester to stir evenly.

[0055] Example 5

[0056] This embodiment discloses a prothrombin time detection or determination kit, which is composed of: 9 g of glycine, 1.8 g of anhydrous calcium chloride, 1 mL of Pc-300, 40 ml of tissue factor ester, and pure water.

[0057] Among them, tissue factor ester is an activated tissue factor made by esterification of recombinant protein (rhTF243) and synthetic phospholipids.

[0058] This embodiment also discloses a method for preparing a prothrombin time detection or determination kit, comprising the following steps: dispersing Pc-300 in pure water, then adding glycine into the system for dispersion, and finally adding anhydrous calcium chloride and recombinant tissue factor ester to stir evenly.

[0059] Example 6

[0060] This embodiment discloses a prothrombin time detection or determination kit, which is composed of: 9 g of glycine, 1.8 g of anhydrous calcium chloride, 40 ml of tissue factor ester, and pure water.

[0061] Among them, tissue factor ester is an activated tissue factor made by esterification of recombinant protein (rhTF243) and synthetic phospholipids.

[0062] This embodiment also discloses a method for preparing a prothrombin time detection or determination kit, comprising the following steps: dispersing glycine in pure water, and then adding anhydrous calcium chloride and recombinant tissue factor ester to mix evenly.

[0063] Comparative Example 1

[0064] The test kit produced by Wuhan Zhongtai was used in this comparative example.

[0065] Experimental Section

[0066] 1. Normal plasma sample values

[0067] 30 human plasma samples with normal prothrombin time (PT) were selected and tested using the freeze-dried formulation and liquid formulation kits prepared in Example 1. Each sample was tested once and the average value of the test was calculated. The results are shown in Tables 1 and 2, respectively: The average value of the normal plasma sample tested by the kit was determined based on the experimental results, and the test results exceeding 14s were considered outliers.

[0068] Table 1 Evaluation data record table of dry powder dosage form test kit for plasma samples

[0069]

[0070]

[0071] Results: From the above data analysis, in the absence of outliers, the PT results of 30 normal human plasma samples were no more than 14s, and the mean value of the reagent test results was 12.44, which met the experimental technical requirements.

[0072] Table 2 Liquid type test kit test sample mean value evaluation data record table

[0073]

[0074]

[0075] Results: From the above data analysis, there were no outliers, the PT results of 30 normal human plasma samples were no more than 14s, and the mean value of the reagent test results was 11.56, which met the experimental technical requirements.

[0076] 2. Precision

[0077] The precision of the freeze-dried and liquid kits prepared in Example 1 was evaluated using PT normal quality control products and abnormal quality control products, as shown in Tables 3 and 4.

[0078] According to the CLSI EP05-A3 method for precision evaluation, the same operator or a group of operators were used on the same instrument, using freeze-dried and liquid reagents to repeat the test 10 times for PT normal quality control and abnormal quality control samples. At the end of the evaluation, there were 10 pairs of normal quality control samples and abnormal quality control samples, that is, 10 test results for normal quality control samples and abnormal quality control samples.

[0079] Table 3. Raw data record of PT normal quality control product test for freeze-dried reagent precision

[0080] Number of detections Quality control product level 1 (unit: s) Quality control product level 2 (unit: s) Quality control product level 3 (unit: s) 1 13.5 28.6 55.0 2 13.2 28.9 55.4 3 13.2 29.0 55.3 4 13.2 29.2 55.4 5 13.2 29.2 55.7 6 13.2 29.2 55.6 7 13.2 29.2 55.6 8 13.2 29.3 55.7 9 13.2 29.2 55.5 10 13.1 29.1 55.6 M 13.22 29.09 55.48 SD 0.10 0.21 0.21 M)±2SD 【13.01，13.43】 【28.67，29.51】 【55.05，55.91】 CV 0.78％ 0.71％ 0.39％

[0081] Table 4 Original data record of liquid reagent precision PT normal quality control product test

[0082]

[0083]

[0084] First, outliers are judged based on (M)±2SD. If the measured value of a single sample exceeds the mean (M)±2SD, it is considered an outlier.

[0085] Judging from the analysis of Tables 5 and 6, there are no outliers.

[0086] Using the results in the table, calculate the precision CV:

[0087] Table 5. Lyophilized reagents: Kit precision test results

[0088] Precision Sample Concentration Lyophilized reagents PT Quality Control Level 1 0.78％ PT Quality Control Level 2 0.71％ PT Quality Control Level 3 0.39％

[0089] Table 6. Liquid reagents: kit precision test results

[0090] Precision Sample Concentration Liquid reagents PT Quality Control Level 1 2.42％ PT Quality Control Level 2 1.30％ PT Quality Control Level 3 0.37％

[0091] The precision CV of freeze-dried reagents and liquid reagents was less than 5%, which met the experimental design requirements.

[0092] 3. Analysis of interfering substances

[0093] The reagent detects plasma samples with added interfering substances and plasma samples without added interfering substances. When the deviation between the test results with added interfering substances and the test results without added interfering substances exceeds ±10%, it is considered that the concentration of the interfering substances at this time will affect the performance of the detection reagent.

[0094] 3.1 Hemoglobin

[0095] Samples containing different concentrations of hemoglobin were tested, including mixed plasma samples with a PT time of about 12 seconds, and the hemoglobin content was 0, 300, 500, 800, and 1000 mg / dL, as shown in Tables 7 and 8. The test results are as follows:

[0096] Table 7 Freeze-dried type: Test results of hemoglobin interference samples

[0097]

[0098] Table 8 Liquid type: Test results of hemoglobin interference samples

[0099]

[0100] Result analysis: Hemoglobin content has positive interference on PT detection. The PT time of normal plasma samples is about 12s. When the relative deviation of the test results of the interference sample exceeds ±10%, the hemoglobin concentration will affect the performance of the test reagent. Therefore, when the hemoglobin is not higher than 1000mg / dL, there is no interference with the test results.

[0101] 3.2 Fat emulsion

[0102] Samples containing fat emulsions of different concentrations were tested, including mixed plasma samples with a PT time of about 12s, and the fat emulsion contents were 0, 200, 400, 600, and 800 mg / dL, respectively, as shown in Tables 9 and 10. The test results are as follows:

[0103] Table 9 Freeze-dried type: Test results of fat emulsion interference samples

[0104]

[0105] Table 10. Liquid type: Test results of fat emulsion interference samples

[0106]

[0107] Result analysis: The PT time of normal plasma samples is about 12s. When the relative deviation of the test results of interfering samples exceeds ±10%, the fat emulsion concentration will affect the performance of the detection reagent. Therefore, when the fat emulsion is not higher than 800mg / dL, there is no interference with the test results.

[0108] 3.3 Bilirubin

[0109] Plasma samples containing different concentrations of bilirubin were tested, including mixed plasma samples with a PT time of about 12 seconds, and the bilirubin content was 5, 10, and 20 mg / dL, respectively, as shown in Tables 11 and 12. The test results are as follows:

[0110] Table 11 Freeze-dried type: Test results of bilirubin interference samples

[0111]

[0112] Table 12 Liquid type: Test results of bilirubin interference samples

[0113]

[0114] Result analysis: The PT time of normal plasma samples is about 12s. When the relative deviation of the test results of interfering samples exceeds ±10%, the bilirubin concentration will affect the performance of the test reagent. Therefore, when the bilirubin is not higher than 20mg / dL, there is no interference with the test results.

[0115] 4. Comparison between Example 1 and Comparative Example 1

[0116] The clinical samples assigned with PT reagent produced by Wuhan Zhongtai were tested using freeze-dried and liquid test kits respectively, and the results were statistically analyzed. For correlation analysis, if k∈[0.80, 1.20] and r>0.975, it is considered to have good consistency with the same product on the market, as shown in Table 13.

[0117] Table 13 Comparison results of freeze-dried reagent and comparative example 1

[0118]

[0119]

[0120]

[0121] The correlation between Comparative Example 1 and Example 1 is as follows Figure 1 After calculation, the freeze-dried reagent had a correlation coefficient of r[0.9935, 0.9961]≥0.975 with the similar reagent produced by Wuhan Zhongtai in the test of 40 clinical samples. The test results were highly positively correlated. The kit has good clinical detection performance, as shown in Table 14.

[0122] Table 14 Comparison of liquid reagents and comparative example 1

[0123]

[0124]

[0125] After calculation, the correlation of liquid reagent in 40 clinical sample tests is shown in Figure 2 , and the correlation coefficient r of the same reagent produced by Wuhan Zhongtai ∈ [0.9963, 0.9971]≥0.975, the test results are highly positively correlated, and the kit has good clinical detection performance.

[0126] 5. Stability

[0127] 5.1 Accelerated Destabilization

[0128] The test kits prepared in Example 1 were stored at 37±1°C and tested every three or four days (3d, 7d and 10d) according to the assessment indicators. The operation was strictly in accordance with the product instructions and compared with the 0d test results (control) to examine the accelerated destruction stability, as shown in Tables 15 and 16.

[0129] 5.2 Stability during shelf life

[0130] The freeze-dried and liquid test kits prepared in Example 1 were stored at 2°C to 8°C, respectively, and tested according to the assessment indicators at different times (0, 6, 12, 18, 24, and 25 months (M)), and the operation was strictly in accordance with the product instructions, and compared with the 0-day test results (control) to examine the stability. (See Tables 17 and 18)

[0131] 5.3 Transport stability

[0132] Therefore, it is designed that in hot summer weather, the freeze-dried and liquid test kits will be placed in a cold chain transport box for transportation (the reagents are concentrated in the middle of the foam box, and the foam box is placed in the turnover box) and sent from Beijing to Chengdu and then returned to Beijing (this test kit was sent from Beijing on July 22, 2022, transported to Chengdu, and transported back to Beijing on July 28, 2022); in severe winter weather, the test kit will be placed in a transport turnover box (cold chain transport foam box) and sent from Beijing to Chengdu and then returned to Beijing (this test kit was sent from Beijing on December 17, 2021, transported to Chengdu, and transported back to Beijing on December 23, 2021). The returned freeze-dried and liquid test kits will be inspected according to the assessment indicators, strictly operated in accordance with the product instructions, and compared with the 0d test results.

[0133] 5.4 Reagent freeze-thaw stability

[0134] The reagent kit was stored at 2℃~8℃. After opening the bottle, the reagent was placed in -20℃ for freeze-thaw cycles 3 times. The freeze-thaw results of the 3 times were tested and compared with the freeze-thaw test results of 0 times (control) to investigate the freeze-thaw stability of the reagent.

[0135] 5.5 Reagent stability after opening

[0136] After opening the liquid and lyophilized test kits, test the normal quality control products and abnormal quality control products three times each. The average of the test results is taken as the initial value (Day 0). Test continuously for 15 days in strict accordance with the product instructions for use. Compare with the test results on Day 0 (control) to examine the stability of the opened bottles when stored at 2°C to 8°C.

[0137] 5.6 On-machine stability

[0138] After opening the bottle / reconstitution, the dry powder and liquid finished test kits were placed in the reagent compartment of the coagulometer, and tested according to the assessment indicators at different times (0, 5, and 7 days). The operation was strictly in accordance with the product instructions, and the results were compared with the 0d test results (control) to examine the in-machine stability, as shown in Tables 15 and 16.

[0139] Table 15 Results of freeze-dried powder accelerated destruction stability test

[0140]

[0141] Table 16 Liquid type accelerated failure stability test results

[0142]

[0143]

[0144] The results show that after the near-expiration and mid-expiration test kits were stored at 37℃±1℃ for 10 days, all assessment indicators met the technical requirements, and all indicators remained unchanged on the 10th day. Therefore, they are stable when stored at 37℃ for 10 days, as shown in Tables 17 and 18.

[0145] Table 17. Results of stability test of lyophilized powder at 2°C to 8°C

[0146]

[0147] Table 18. Liquid type stability test results at 2℃~8℃

[0148]

[0149]

[0150] It can be seen from Tables 17 and 18 that the freeze-dried powder and liquid type still have good stability when stored at 2°C to 8°C for 25 months, and all evaluation indicators meet the technical requirements.

[0151] 5.3 Transport stability

[0152] Table 19 Transport stability (lyophilized powder) test results (winter)

[0153]

[0154]

[0155] Table 20 Transport stability (liquid type) test results (winter)

[0156]

[0157] Table 21 Transport stability (lyophilized powder) test results (summer)

[0158]

[0159] Table 22 Transport stability (liquid type) test results (summer)

[0160]

[0161]

[0162] The results show that during the transportation process, all evaluation indicators of the liquid and lyophilized test kits prepared in Example 1 meet the technical requirements.

[0163] Table 23 Freeze-thaw stability (lyophilized powder) experimental results

[0164]

[0165] Table 24 Freeze-thaw stability (liquid type) experimental results

[0166]

[0167]

[0168] The results showed that the relative deviation of the test results after three freeze-thaw cycles of the reagent was ≤15%, so the freeze-thaw cycles should not exceed 3 times.

[0169] Table 25 Test results of reagent opening stability (lyophilized powder)

[0170]

[0171] Table 26 Test results of reagent bottle opening stability (liquid type)

[0172]

[0173] The results show that 15 days after the reagent was opened, the relative deviation of the normal / abnormal quality control results and the 0-day test results was ≤10%, the repeatability was ≤5%, and the test results on the 15th day did not change significantly. Therefore, the reagent is stable within 15 days after opening the bottle, which meets the experimental requirements.

[0174] Table 27 In-machine stability (lyophilized powder) experimental results

[0175]

[0176] Table 28 On-machine stability (liquid type) test results

[0177]

[0178] The results showed that there was no significant change in quality control after the dry powder reagent was reconstituted or the liquid reagent was placed on the instrument at 2℃-8℃ for 0, 5, and 7 days.

[0179] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0180] In the present application, in the absence of any contrary description, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present specification, the terms "including", "comprising", etc. refer to "including but not limited to". In this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of the associated objects, indicating that there may be three relationships, for example, A and / or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In this article, "at least one" refers to one or more, and "plural" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of singular or plural items. For example, "at least one of a, b, or c", or "at least one of a, b and c", can both mean: a, b, c, ab (i.e. a and b), ac, bc or abc, where a, b, c can be single or plural, respectively.

[0181] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A prothrombin time detection or determination kit, It is characterized in that The invention comprises the following components in percentage by mass: 3-5% of recombinant tissue factor ester, 0.7-1.2% of stabilizer, 0.08-0.15% of preservative, and the balance is solvent.

2. The prothrombin time detection or determination kit according to claim 1, It is characterized in that The stabilizer includes bovine serum albumin.

3. The prothrombin time detection or determination kit according to claim 1, It is characterized in that The preservatives include Pc-300.

4. The prothrombin time detection or determination kit according to any one of claims 1 to 3, It is characterized in that The kit also includes glycine with a mass percentage of 7-9%.

5. The prothrombin time detection or determination kit according to claim 4, It is characterized in that The kit also includes anhydrous calcium chloride with a mass percentage of 1.5-2.0%.

6. The prothrombin time detection or determination kit according to claim 1, It is characterized in that The kit of 1L volume includes the following components: 9g of glycine, 1.8g of anhydrous calcium chloride, 0.8g of bovine serum albumin, 1mL of Pc-300, 40ml of tissue factor ester, and the rest is pure water.

7. The prothrombin time detection or determination kit according to claim 1 or 6, It is characterized in that The recombinant tissue factor ester is obtained by esterifying a recombinant protein and a synthetic phospholipid, and the recombinant protein is rhTF243.

8. The prothrombin time detection or determination kit according to claim 1, It is characterized in that The dosage form of the reagents in the kit is a lyophilized dosage form or a liquid dosage form.

9. A method for preparing a prothrombin time detection or determination kit according to any one of claims 1 to 7, It is characterized in that The stabilizer and preservative are dispersed in the solvent, and then the recombinant tissue factor ester is added thereto and stirred evenly.

10. Use of the prothrombin time detection or determination kit according to any one of claims 1 to 8 in coagulation detection.

Images