Method for determining thrombin-like enzyme activity

A technology similar to thrombin and activity, applied in the field of medical testing, can solve the problems of subjective interference of the tester, long time consumption, linearity, sensitivity, poor accuracy, etc., achieve the effect of simple and unified reagent source, simple operation process, and avoid reagent manufacturers

Active Publication Date: 2014-06-25
GRAND LIFE SCI (LIAONING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for these two methods, both plasma and fibrinogen have complex sources, and the differences between different manufacturers are large. Even if the products of the same manufacturer are different, the differences between different batches are also large, and the storage time of the two methods will also affect the activity determination. , not to mention the result of visual judgment, subject to greater subjective interference
[0007] Hefei Zhaoke Pharmaceutical Co., Ltd. has applied for the determination method of batroxobin activity. Defibrase is used as a standard product to measure batroxobin. The defibrase activity Bu is the batroxobin activity unit. The determination method adopts the traditional enzyme fixation time Determination method, this type of method is not only time-consuming, but also difficult to ensure that the reaction is a zero-order reaction (the reaction speed is directly proportional to the activity of the tested enzyme, and has nothing to do with the concentration of the substrate), so its linearity, sensitivity, and accuracy are poor

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  • Method for determining thrombin-like enzyme activity
  • Method for determining thrombin-like enzyme activity
  • Method for determining thrombin-like enzyme activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 The selection of standing time and reaction time in assay method of the present invention

[0039] In this example, the recombinant site-directed mutant batroxobin (ZL200710011566.4) was used as the thrombin-like sample to be tested, and the resting time and reaction time in the activity determination method were screened.

[0040] Prepare 2mmol / L S-2238 (chromogenix, Italy) substrate aqueous solution with deionized water. The recombinant site-directed mutation batroxobin with batch number 20100101 produced by Nuokang Biotechnology Co., Ltd. was randomly selected as the test sample, and the sample was diluted to 100 nmol / L with 50 mmol / L Tris-HCl pH7.4.

[0041] Add 60 μL recombinant batroxobin dilution solution for site-directed mutagenesis, 890 μL 50 mmol / L Tris-HCl, and 50 μL 2 mmol / L S2238 aqueous solution into a 1 mL cuvette, and mix well. Put the cuvette into a 37°C UV spectrophotometer (thermo, evolution220, USA), and immediately measure the absorban...

Embodiment 2

[0046] Example 2 The selection of substrate in assay method of the present invention

[0047] In this example, the recombinant site-directed mutant batroxobin (ZL200710011566.4) was used as the thrombin-like sample to be tested, and the substrates in the activity assay method were screened.

[0048] Prepare 2 mmol / L aqueous solutions of S2238, S2288, S2251, S2366, S2403, and S2765 substrates with deionized water. The recombinant site-directed mutation batroxobin with batch number 20100101 produced by Nuokang Biotechnology Co., Ltd. was randomly selected as the test sample, and the sample was diluted to 100 nmol / L with 50 mmol / L Tris-HCl pH7.4.

[0049] Add recombinant site-directed mutagenesis batroxobin dilution solution to 1mL cuvette, add 50 μL of the above-mentioned 2mmol / L S2238, S2288, S2251, S2366, S2403, S2765 substrate aqueous solution, then supplement the Tris-HCl to 1mL, mix well, The batroxobin had a final concentration of 1-17 nmol / L, stood at 37° C. for 5 minu...

Embodiment 3

[0051] Example 3 Selection of the linear range of the final concentration of the enzyme sample to be tested in the assay method of the present invention

[0052] In this example, the recombinant site-directed mutant batroxobin (ZL200710011566.4) was used as the thrombin-like sample to be tested, and the linear range of the final concentration in the activity determination method was screened.

[0053] The recombinant site-directed mutation batroxobin enzyme with batch number 20100101 produced by Nuokang Biotechnology Co., Ltd. was randomly selected as a test sample, and diluted to 100 nmol / L with 50 mmol / L Tris-HCl pH7.4. Add different volumes of the enzyme dilution solution to be tested, Tris-HCl buffer and chromogenic substrate to obtain a 1 mL reaction system, as shown in Table 1.

[0054] Table 1 1mL reaction system with different compositions

[0055]

[0056]

[0057] Put the above different reaction systems into a UV spectrophotometer at 37°C and let it stand f...

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Abstract

The invention relates to a method for determining a snake venom thrombin-like enzyme. An enzyme kinetics determination method is adopted, a controlled temperature ultraviolet spectrophotometer is used, and the incubating time, the enzyme concentration, the linear range and the accuracy and enzymatic reaction kinetics constants of the method are investigated, so as to finally determine the determination method of the thrombin-like enzyme activity. By adopting the method, the adverse factors that the traditional method is large in performance difference of raw materials such as plasma or fibrinogen and the like for detection, and large in subjective effects of operating personnel, and just can be used for determining by a specially-assigned person are overcome, the method has the advantages of being simple in step, clear in experiment data, and accurate and reliable in determination result, does not need to draw a standard curve, is applicable to activity determination, activity definition and quality standard control of thrombin-like enzyme stoste and preparations with various sources, including natural and recombinant thrombin.

Description

technical field [0001] The invention belongs to the technical field of medical detection, in particular, the invention relates to a method for measuring thrombin-like activity. Background technique [0002] Since 1936, Austrian scholar Von Klobusitzky first isolated a serine proteolytic enzyme (thrombin-like enzyme) from the venom of the Brazilian spearhead viper (Bothropsatrox), namely batroxobin, and more than 30 kinds of snake venoms have been found to contain thrombin-like groups. points, and more than 20 kinds have been separated and purified successively. [0003] Most of the snake venom preparations currently used clinically in my country are thrombin-like, which have two clinical uses. [0004] One is the defibrinating effect, that is, batroxoblastase, such as Dongling Difu, whose function is to decompose fibrinogen and inhibit thrombus formation; induce the release of tissue plasminogen activator (t-PA), Weaken the activity of inhibitory factor of plasminogen acti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N1/28G01N1/38
Inventor 徐梅李娜李秀娜石皎薛雁王宏英薛百忠
Owner GRAND LIFE SCI (LIAONING) CO LTD
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