Quantitative detection method of venom thrombin-like enzyme

A quantitative detection method and thrombin-like technology, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, measuring devices, etc., can solve the problems of cumbersome determination methods, poor accuracy, and the inability to accurately characterize the quality of snake venom, and achieve improvement. The level of quality control, quantitative accuracy, and the effect of filling the gaps in quality standards

Active Publication Date: 2020-11-06
SHANDONG INST FOR FOOD & DRUG CONTROL +2
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  • Application Information

AI Technical Summary

Problems solved by technology

The currently reported methods for determining the content of thrombin-like enzymes in snake venom are mainly gel electrophoresis and potency assay.

Method used

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  • Quantitative detection method of venom thrombin-like enzyme
  • Quantitative detection method of venom thrombin-like enzyme
  • Quantitative detection method of venom thrombin-like enzyme

Examples

Experimental program
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preparation example Construction

[0031] The preparation method of relevant reagent reagent and solution in the following examples is as follows:

[0032] (1) Reagents: trypsin (Sigma, batch number SLBS8956), Agkistrodon haemagglutinate (Jinzhou Aohong Pharmaceutical Co., Ltd., purity 98.5%), guanidine hydrochloride (VETEC, batch number WXBC4261V), trihydroxyaminomethane (Shanghai Stock Exchange , batch number 20181206), dithiothreitol (BBI Life Sciences, batch number D911BA0011), iodoacetamide (BBI Life Sciences, batch number B326BA1943), and other reagents were of analytical grade.

[0033] (2) 25 mmol / L ammonium bicarbonate solution: Weigh 79.06 mg of ammonium bicarbonate, add 40 mL of water to dissolve, and obtain.

[0034] (3) Dithiothreitol (DTT) solution: Weigh 15.42 mg of dithiothreitol and add 500 μL of water to dissolve it.

[0035] (4) Iodoacetamide (IA) solution (newly prepared before use): Weigh 18.5 mg of iodoacetamide, add 500 μL of water to dissolve, and obtain the solution.

[0036] (5) 0.4 ...

Embodiment 1

[0038] Screening and Determination of Characteristic Peptides of Thrombin in Agkistrodon halys

[0039] 1 Instruments and equipment

[0040] Thermo Fusion high-resolution mass spectrometer (Thermo Fisher Scientific, USA), EASY-nLC 1000 nanoliter liquid chromatograph (Thermo Fisher Scientific, USA), CP225D electronic balance (Sartorius, Germany), Sigma 3-30 K refrigerated centrifuge ( Sigma, Germany), Milli-QAdvantage A10 ultrapure water meter (Millipore, USA).

[0041] 2 Chromatography and mass spectrometry conditions

[0042] Chromatographic column: 0.2 mm × 3.5 cm (5 μm particle size) ReproSil-Pur C made in the laboratory 18-AQ Trapcolumn desalted and enriched, using a laboratory-made 75 μm × 25 cm (3 μm particle size) ReproSil-Pur C18-AQ nanoliter analysis column for separation. Mobile phase A was 2% acetonitrile in 0.1% formic acid in water; mobile phase B was 98% acetonitrile in 0.1% formic acid in water. The flow rate of the nanoliter separation pump is 300 nL / min, a...

Embodiment 2

[0050] Multiple reaction monitoring (MRM) was used to quantify the thrombin in the samples of Agkistrodon halys.

[0051] 1. Instruments and equipment

[0052] SCIEX Triple Quad 6500 triple quadrupole mass spectrometer, CP225D electronic balance (Sartorius, Germany), Sigma 3-30 K refrigerated centrifuge (Sigma, Germany), Milli-QAdvantage A10 ultrapure water instrument (Millipore, USA).

[0053] 2. Chromatography and mass spectrometry conditions

[0054] Liquid phase conditions: Waters ACQUITY UPLC BEH C 18 Chromatographic column (50 mm×2.1 mm, 1.7 μm); column temperature: 40°C; injection volume: 2 μL; flow rate: 0.2 mL / min; mobile phase A is 0.1% formic acid, B is 0.1% formic acid in acetonitrile, Gradient elution, elution program: 0→1 min, mobile phase A 80%; 1→5 min, mobile phase A 80%→10%; 5→7 min, mobile phase A 10%→10%.

[0055] Mass spectrometry conditions: electrospray ion source, positive ion scanning mode, multiple reaction monitoring; vortex ion spray temperature...

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Abstract

The invention relates to the technical field of chemical analysis and quantitative detection, in particular to a quantitative detection method of venom thrombin-like enzyme. The quantitative detectionmethod of the venom thrombin-like enzyme comprises the following steps: taking an agkistrodon halys venom thrombin-like enzyme characteristic polypeptide reference substance with an amino acid sequence of LDSPVSNSAHPLSLPSSAPSVGSVCR to prepare a series of concentration reference substance solutions; respectively adding the test solution, carrying out enzymolysis, and taking the supernatant after enzymolysis as a series of solutions to be detected; and respectively injecting a to-be-detected solution into a liquid chromatography-mass spectrometer, and selecting a qualitative ion pair and a quantitative ion pair to detect the content of the characteristic polypeptide in the to-be-detected solution. According to the method, the content of the thrombin-like enzyme in the agkistrodon halys venom and the extract thereof is determined by adopting a standard addition method, the method is simple, convenient and rapid, the matrix interference is less, the sensitivity is high, the quantificationis accurate, the blank of the quality standard of the agkistrodon halys venom and the extract thereof is filled, and the quality control level is improved.

Description

technical field [0001] The invention relates to the technical field of chemical analysis quantitative detection, in particular to a quantitative detection method of snake venom thrombin. Background technique [0002] Snake venom-like thrombin is a glycoprotein extracted and separated from snake venom, which has the functions of reducing plasma fibrinogen and blood viscosity. It has been used as a drug for the treatment of thrombotic diseases for more than 50 years. In addition to the treatment of cerebral embolism, thromboangiitis obliterans, femoral artery embolism, pulmonary embolism and other thrombotic diseases and prevention of postoperative thrombosis recurrence, it has certain curative effect on kidney disease, lupus erythematosus, viral hepatitis and Raynaud's disease. [0003] The white-browed Agkistrodon halys is a unique species in my country, mainly distributed in the Changbai Mountains in Northeast my country. The venom of Agkistrodon halys is complex, containi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72
CPCG01N30/02G01N30/06G01N30/72C12Q1/37G01N2333/96422G01N33/6848G01N30/7266G01N30/8624G01N2030/027G01N2030/067
Inventor 石峰咸瑞卿巩丽萍迟连利张群业杜宏明王聪聪
Owner SHANDONG INST FOR FOOD & DRUG CONTROL
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