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Fibrinogen plate assay for detection of thrombin, thrombin-like enzyme and thrombin inhibitor

A thrombin inhibitor and fibrinogen technology, applied in the field of biological protease, can solve the problems of turbidity or color interference, large difference in freezing point, low sensitivity and other problems, and achieve easy promotion and use, low cost and high sensitivity high effect

Inactive Publication Date: 2006-04-26
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are large differences in the freezing point (ie, the end point of the reaction) judged by the naked eye, mutual interference of turbidity or color of the test product, low sensitivity (10 enzyme activity units per ml), inaccurate quantification, etc.
With the artificially synthesized chromogenic substrate, the quantitative detection of thrombin was realized with the thrombin chromogenic substrate method, and its sensitivity was greatly improved (2.5 enzyme activity units per milliliter were detected); but due to the chromogenic substrate The reagents are too expensive, the equipment conditions are high during the determination, and the turbidity or color of the sample is also easily interfered, so its application is limited
This brings certain difficulties to the in-depth research, development and utilization of thrombin, thrombin inhibitors, thrombin-like enzymes (defibrase from snake venom), etc.

Method used

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  • Fibrinogen plate assay for detection of thrombin, thrombin-like enzyme and thrombin inhibitor
  • Fibrinogen plate assay for detection of thrombin, thrombin-like enzyme and thrombin inhibitor
  • Fibrinogen plate assay for detection of thrombin, thrombin-like enzyme and thrombin inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Quantitative detection of thrombin

[0027] ①Preparation of 2.5% agarose: Weigh 2.5 grams of agarose, put it in a flask, add distilled water to 100ml, and set it in a 100°C water bath for about 30 minutes to fully dissolve the agarose for later use.

[0028] ②Prepare 0.1% (percent concentration, the same below) fibrinogen buffer solution: Weigh 8 mg of human fibrinogen and dissolve it with 8 ml of 0.1M pH7.4 phosphate buffer.

[0029] ③ Plate making: Take a flat dish with a diameter of 9 cm and place it on a level table (measured with a spirit level). Heat the prepared 2.5% agarose to 90-100°C, mix 8 ml with 8 ml of 0.1% fibrinogen buffer, and pour it into a plate immediately. After cooling at room temperature, it is a fibrinogen plate.

[0030] ④ Punching: Punch holes at 1.6 cm intervals on the cooled fibrinogen agar plate. The aperture is 4 mm.

[0031] ⑤Preparation and sample addition reaction of standard and test products: dissolve the standard with 0....

Embodiment 2

[0034] Example 2: Quantitative detection of thrombin

[0035] Steps ① to ④ are the same as in Example 1.

[0036] ⑤Preparation and sample addition reaction of standard and test products: dissolve the standard with 0.05M pH7.4 phosphate buffer, and dilute to 5.0U / ml, 2.5U / ml, 1.25U / ml, 0.62U / ml, 0.31 U / ml, 0.15U / ml series concentration. Dissolve the test sample in 1 ml of the same buffer and make a 160-fold dilution. Add the diluted standard substance and test substance to the above-mentioned plate wells, and add 18 μl of sample to each well. The plate was covered and then placed at 37°C for a constant temperature diffusion reaction for 7 hours.

[0037] ⑥Draw the standard curve, calculate the result: use the oil caliper to measure the diameter of the sedimentation circle of each reaction hole, the standard hole: in the order of 5→0.15U / ml, the diameters of the sedimentation circle are 7.94mm, 7.62mm, 7.20mm, 6.78mm. , 6.24mm, 5.94mm;

[0038]Test hole: diluted 160 times t...

Embodiment 3

[0039] Example 3: Quantitative detection of thrombin-like enzymes

[0040] ① Same as Example 1.

[0041] ②Prepare 0.15% fibrinogen buffer: Weigh 12mg of human fibrinogen and dissolve it in 8ml of 0.1M pH7.4 phosphate buffer.

[0042] ③ Plate making: Take a plate with a diameter of 9 cm and place it on a horizontal table (measured with a level ruler). Heat the prepared 2.5% agarose to 90-100°C, take 8ml and mix with 8ml of 0.15% fibrinogen buffer, pour it into a plate immediately, and after cooling at room temperature, it becomes a fibrinogen plate.

[0043] 4. punching: with embodiment 1.

[0044] ⑤Preparation of standard and inspection products and sample addition reaction: dissolve the standard with 0.05M PH7.4 phosphate buffer, and dilute to 1.25U / ml, 0.62U / ml, 0.31U / ml, 0.15U / ml, 0.075 U / ml serial concentration. Dissolve the sample with 1ml of the same buffer and make a 10-fold dilution. Add the diluted standard substance and test substance to the wells of the above-m...

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Abstract

The fibrinogen plate method for detecting thrombase, batroxobin and thrombase inhibitor includes preparing fibrinogen plate with agarose and fibrinogen and quantitative detection via the diffusion reaction of standard sample and detected sample on the plate. The detection method of the present invention has simple operation, high sensitivity, being intuitive and stable, no interference of the turbidity and color of the detected sample and low cost. The detection method may be realized simply in lab with thermotank.

Description

technical field [0001] The present invention belongs to the field of biological protease, particularly thrombin, thrombin-like and thrombin inhibitor detection methods. Background technique [0002] Thrombin, thrombin-like and thrombin inhibitors (such as hirudin, heparin) widely exist in animals and plants, and are the main research objects in medicine, bioengineering and biopharmaceuticals. Determination of biological activity of thrombin, thrombin-like enzymes and their inhibitors is a necessary condition for scientific research and industrial production. There are two main established biological activity detection methods: thrombin titration method and thrombin chromogenic substrate method. Thrombin titration is the most widely used because of its simple and rapid operation. However, there are problems such as large differences in judging the freezing point (ie reaction end point) with the naked eye, mutual interference between the turbidity or ...

Claims

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Application Information

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IPC IPC(8): C12Q1/37C12Q1/56G01N33/52G01N33/68
Inventor 廖共山班建东
Owner GUANGXI MEDICAL UNIVERSITY
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