Method for calibrating activity of coagulation factor X activator

A blood coagulation factor and activator technology, which is applied in the field of biological product calibration and blood coagulation factor X activator activity calibration, can solve the problem that the pH of the buffer solution cannot be kept stable, and achieve the reduction of calibration cost, strong buffer capacity, and good precision of reaction rate Effect

Active Publication Date: 2018-05-29
STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After repeated experiments, the ratio of suitable substrate to enzyme was obtained; secondly, the pH of the buffer could not be kept stable

Method used

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  • Method for calibrating activity of coagulation factor X activator
  • Method for calibrating activity of coagulation factor X activator
  • Method for calibrating activity of coagulation factor X activator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Activity calibration of blood coagulation factor X activator

[0050] 1. Preparation of reagents used in the examples

[0051] (1) Preparation of 200mM Tris-HCl buffer solution (pH7.4)

[0052] Accurately weigh 24.2g Tris, add 800mL of water to dissolve, cool to room temperature, adjust the pH value to 7.4 with 1mol / L HCl, add water to 1000mL, mix well and set aside.

[0053] (2) 75mM CaCl 2 prepare

[0054] Weigh 0.366g CaCl 2 , add distilled water to dissolve, cool to room temperature, add water to 100ml, mix well and set aside.

[0055] (3) Preparation of 200mmol / L Tris-HCl buffer solution (pH7.4) containing 0.1% human serum albumin

[0056] Take 100 μl of 20% human serum albumin injection, add 200 mmol / L Tris-HCl buffer solution (pH 7.4) to 20 ml, mix well, and set aside.

[0057] (4) Preparation of 25μg / ml blood coagulation factor X

[0058] Add 1mL of water to the reagent bottle of coagulation factor X (manufacturer: HYPHEN BioMed, product number:...

Embodiment 2

[0070] Embodiment 2 method verification

[0071] Adopt the method operation of " above-mentioned embodiment 1 ". The following verification results are obtained:

[0072] 1 precision

[0073] (1) In-board precision

[0074] Samples (refer to literature Hong-Sen Chen, Jin-Mei Chen, Chia-Wei Lin, etc. Newinsights into the functions and N-glycan structures of factor X activator from Russell's viper venom. FEBS[J].275(2008)3944– 3958) were formulated into low, medium and high concentration samples (0.05, 0.1, 0.2U / ml), and the repeatability was evaluated by 10 repeated experiments.

[0075] Table 1 In-panel precision inspection results

[0076]

[0077] It can be seen from the results in Table 1 that the test results at low, medium and high concentrations all have good in-panel precision, and the in-panel precision RSD<5%.

[0078] (2) Intermediate precision

[0079] Table 2 The results of intermediate precision investigation

[0080]

[0081] It can be seen from the ...

Embodiment 3

[0133] Embodiment 3, blood coagulation factor X activator activity detection method

[0134] 1. Preparation of reagents used in the examples

[0135] Reagent 75mM CaCl used in this embodiment 2 , 0.1% human serum albumin 200mmol / L Tris-HCl buffer solution (pH7.4), 25 μg / ml blood coagulation factor X, chromogenic substrate solution, concrete preparation is with the reagent used in embodiment 1 and preparation method.

[0136] Standard product preparation: the standard product obtained by the calibration method in Example 1, that is, take one standard product of 14U / cartridge, and dilute it with Tris-HCl buffer solution to 5 concentrations of 0.025, 0.05, 0.1, 0.2, 0.4U / mL for later use.

[0137] Sample preparation to be tested: refer to the literature Hong-Sen Chen, Jin-Mei Chen, Chia-Wei Lin, etc. New insights into the functions and N-glycan structures of factor X activator from Russell's viper venom. FEBS[J].275(2008) 3944-3958 for preparation, and then dilute the resulting...

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Abstract

The invention discloses a method for calibrating activity of a coagulation factor X activator and belongs to the field of biotechnology. The steps are as follows: (1) adding coagulation factor X witha concentration of 22.5 mug/ml to 27.5 mug/ml, a chromogenic substrate solution, and a to-be-calibrated sample with a determined dilution ratio into an enzyme linked immunosorbent assay (ELISA) plate;wherein the chromogenic substrate solution is a solution obtained by evenly mixing a chromogenic substrate solution with the concentration of 4.5 mg/ml to 5.5 mg/ml with a CaCl 2 solution in a volumeratio of 4:3, the volume ratio of the coagulation factor X to the chromogenic substrate solution to the to-be-calibrated sample is 30:23:7, and at least 2 parallel ways are arranged in the same sample; (2) putting the ELISA plate into an ELISA instrument for reading of OD value; (3) taking the OD value of the reaction period of 4 to 15 minutes, and performing linear regression on the average OD value of the parallel ways and the square of the reaction time to obtain a reaction curve; and (4) selecting the slope of the reaction curve, defining the activity unit, and multiplying the dilution ratio of the to-be-calibrated sample to obtain the activity of the to-be-calibrated sample. The method has the advantages of simple operation, stable reaction rate, high calibration precision and high accuracy.

Description

technical field [0001] The invention relates to a method for calibrating biological products, in particular to a method for calibrating the activity of coagulation factor X activators, and belongs to the field of biotechnology. Background technique [0002] Coagulation factor X activator is an enzyme with proteolytic properties that can act on coagulation factor X and coagulation factor IX. So far, the earliest and most active activator of blood coagulation factor X was discovered from the venom of the round-spotted viper (Vipera russelli), and the English abbreviation is RVV-X. This is a glycoprotein with a relative molecular mass of 92880 and contains one heavy chain and two light chains. It specifically cleaves the peptide bond between Arg-Ile at position 194 in blood coagulation factor X, and converts the inactive blood coagulation factor X (FactorX) is converted into enzymatically active coagulation factor X a (Factor X a ), thereby exerting a procoagulant effect. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/50
CPCG01N33/5008G01N33/68G01N2333/96444
Inventor 郝鹏飞冯彩丽谭淑萍
Owner STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
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