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Thrombin-like enzyme titer determination method

A titer determination and thrombin-like technology, which is applied in the field of hemocoagulase determination, can solve problems such as product titer differences and the impact of product titer consistency, and achieve accurate measurement results

Active Publication Date: 2013-09-18
GRAND LIFE SCI (LIAONING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the potency determination of commercially available thrombin-like products is carried out by visual inspection. However, due to the different standards for judging the coagulation end point by human eyes in this method, there are differences in potency between products, and the method Human-citrate plasma is widely used as the substrate in the determination, and there are some differences in the human-citrate plasma produced by different blood centers, which has a certain impact on the consistency of product titer

Method used

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  • Thrombin-like enzyme titer determination method
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  • Thrombin-like enzyme titer determination method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The mensuration of embodiment 1 class thrombin

[0022] Analysis steps:

[0023] 1) Turn on the hemagglutination analyzer and wait for the temperature to reach 37°C for standby;

[0024] 2) After re-dissolving the standard product, dilute it with diluent to obtain thrombin-like standard products with concentrations of 2.5U / ml, 2.0U / ml, 1.3U / ml, 1.0U / ml, 0.7U / ml and 0.4U / ml ;

[0025] 3) Take 0.1ml coagulation quality control plasma and add it to the sample tube and place it in the detection part of the coagulation analyzer to preheat for 2 minutes;

[0026] 4) Precisely measure 0.2ml of standard solution, add it to each sample tube, close the pipette stand, and measure;

[0027] 5) record the coagulation time;

[0028] 6) Interpretation of results: ① Calculate the logarithm of the time base 10 and the logarithm of the concentration base 10. ② Take the logarithm of time as the ordinate and the logarithm of the concentration as the abscissa, and make a standard curve...

Embodiment 2

[0032] Example 2 Verification of the methodology for measuring thrombin-like titer

[0033] 1) Standard curve

[0034] According to the operating method of Example 1, the standard curve was determined three times, and the results are shown in Table 1 and Table 2.

[0035] Table 1

[0036]

[0037] Table 2

[0038]

[0039] The above experiment shows that the repeatability of the results is good, and the linear relationship of the thrombin-like standard curve is above 0.99, which has a good linear relationship and meets the needs of the determination.

[0040] 2) Accuracy

[0041] Take thrombin-like titer standard product, batch number: 20110512, specification: 100U / cartridge, dilute with diluent to concentration 0.4, 0.5, 0.6, 0.8, 1.0, 1.2, 1.6, 2.0, 2.4 units / ml solution, as above Method detection, record test data, calculate recovery rate, the results are shown in Table 3, 4, 5.

[0042] table 3

[0043]

[0044] Table 4

[0045]

[0046] The above experi...

Embodiment 3

[0057] Example 3 comparison of thrombin titer assay methods

[0058] 1) Determination of thrombin-like titer in human plasma

[0059] Precisely measure 0.2ml of human plasma, put it in a small test tube, keep it warm at 37±0.5 for 3min, add 0.2ml of the test solution, shake it up immediately, and time it at the same time, record the amount of enzyme whose initial setting time is 60±20s as 1 unit.

[0060] Take thrombin-like enzyme, batch number 110408, and after diluting it 1000 times, different people will measure it according to the above method. The results are shown in Table 8.

[0061] Table 8

[0062]

[0063] Take thrombin-like enzyme, batch number 110408, and after diluting 1000 times, different batches of human-citrated plasma were measured according to the above method. The results are shown in Table 9.

[0064] Table 9

[0065]

[0066] 2) Determination of thrombin-like titer in quality control plasma

[0067] According to the operation method of Example...

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Abstract

The invention provides a thrombin-like enzyme titer determination method. According to the invention, blood coagulation quality control plasma is adopted as a substrate, and determination is carried out by using a blood coagulation analyzer. With the thrombin-like enzyme, fibrinogen in the plasma can be directly hydrolyzed into fibrin, such that the plasma is coagulated. With a principle that plasma coagulation time is inversely proportional to the thrombin-like enzyme titer within a certain thrombin-like enzyme concentration range, the titer of the thrombin-like enzyme is obtained. With the method provided by the invention, product titer differences caused by different criteria of surveyors upon coagulation terminal points and differences in human-citric acid plasma are avoided. Therefore, highly efficient and accurate determinations upon thrombin-like enzyme are realized.

Description

technical field [0001] The invention belongs to the field of determination of hemocoagulase, in particular to a method for determining the titer of thrombin-like enzymes. Background technique [0002] In 1936, Klobusizky and Konig isolated an enzyme protein that can shorten the coagulation time from the venom of the willow-leaf viper, named Coagulase, and many scholars successively confirmed the existence of this enzyme in different Vipers of the Viper family. Since the 1960s, with the improvement of protein purification and separation technology and the in-depth research of biochemistry and pharmacology, the huge cost of thrombin-like application in clinical hemostasis has been determined. Thrombin-like is a polypeptide single-chain thrombin-like hemostatic agent with a molecular weight of about 36000Da. It has many advantages such as high efficiency, long-term effect, safety and convenient use, and is not affected by the inhibition of plasma thrombin. It is often used fo...

Claims

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Application Information

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IPC IPC(8): C12Q1/56
Inventor 薛百忠王宏英孙东崔亮亮李秀琳李秀娜薛雁沈文
Owner GRAND LIFE SCI (LIAONING) CO LTD
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