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Method for measuring activity of enzyme for dissolving thrombus

A technology of activity measurement and measurement method, which is applied in the field of determination of thrombolytic enzyme activity, can solve the problems of cumbersome production of thrombus, low measurement efficiency, obsolete and backwardness, etc., achieve shortened measurement time, improve measurement efficiency, and high precision and accuracy Effect

Inactive Publication Date: 2007-02-28
NANKAI UNIV
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Problems solved by technology

Sidelmann et al. added thrombin and fibrinogen into the small holes of the microtiter plate, and after 4 hours of coagulation, added the plasmin solution to be tested in the holes, let it stand at 25°C for 17 hours, and measured thrombus at 405nm Absorbance value, this method is outdated and backward, and the determination efficiency is low
Fossum et al. added o-nitroaniline to increase the sensitivity of the absorbance value at 405nm when making microthrombosis. However, the thrombus production in this article is cumbersome, and it only takes one day to make the thrombus. After adding thrombolytic enzyme, it needs to stand still at 22°C for 17 Hours, the absorbance value of the thrombus was measured at 405nm, which is also time-consuming
[0004] About adding agarose to the system when making microthrombosis, so that it can form a uniform thrombus within 1 hour, and after adding thrombolytic enzyme solution, use 37 ° C shaking incubation, so as to complete the determination of thrombolytic enzyme within 2 hours The method has not been reported in the literature

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  • Method for measuring activity of enzyme for dissolving thrombus
  • Method for measuring activity of enzyme for dissolving thrombus
  • Method for measuring activity of enzyme for dissolving thrombus

Examples

Experimental program
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experiment Embodiment 1

[0021] Experimental Example 1: Urokinase Standard Curve Determination 1

[0022] To make microthrombosis: use 0.04mol / L, pH7.8 sodium barbital buffer solution to prepare fibrinogen solution with a concentration of 6.7mg / mL, a thrombin solution with a concentration of 300u / mL, and a plasma solution with a concentration of 1 casein unit / mL. Enzyme solution, 1.28% (w / v) agarose solution, add thrombin solution to the agarose solution so that the final concentration of thrombin is 2.6u / mL, the specific operations are as follows:

[0023] Take a 50ml test tube, add 2.5ml of agarose solution to the test tube, place the test tube in a water bath at 40-42°C, add 35 μL of thrombin solution to the test tube, add 50 μL of plasma lysinogen solution of 1 casein unit / mL , shake and mix well, then add 1.5 mL of fibrinogen solution, mix quickly and add 150 μL to the small well of the cell culture plate, and incubate the plate in a humid box at 37°C for 1 hour to make a microthrombosis plate. ...

experiment Embodiment 2

[0034] Experimental Example 2: Urokinase Standard Curve Determination 2

[0035] When making microthrombosis, the concentration of the agarose solution was prepared to be 1.6%, and the rest of the steps were the same as in Experimental Example 1. Figure 3 urokinase standard curve can be made, the standard curve fitting equation is y=0.022x+1.3656, r 2 =0.9904

experiment Embodiment 3

[0036] Experimental Example 3: Urokinase Standard Curve Determination 3

[0037] When making microthrombosis, the concentration of the agarose solution was prepared to be 0.96%, and the rest of the steps were the same as in Experimental Example 1. Figure 4 urokinase standard curve can be made, the standard curve fitting equation is y=0.0208x+1.3648, r 2 =0.9801

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Abstract

A process for testing the activity of thrombus lysozyme includes preparing the miniature thrombus plate by adding agarose to the thrombus forming system prepared from fibrinogen solution and thrombase solution, adding thrombus lysozyme solution to the hole of miniature thrombus, shaking at 37 deg.C while measuring the optical absorbency at 630 nm every a time interval, and calculating the variation of optical absorbency.

Description

technical field [0001] The invention relates to a method for measuring thrombolytic enzyme activity, in particular, a method for measuring thrombolytic enzyme activity using a microthrombosis plate. Background technique [0002] In 1952, Tage Astrup and Sten Müllertz established a fibrin plate method for the determination of thrombolytic enzyme activity. In this experiment, a thrombus plate is made in a plate, and then a hole is punched on the plate, and a thrombolytic enzyme solution is injected into the hole, and after incubation at 37°C for 18-20 hours, the diameter of the dissolved circle is measured, and the product of the vertical diameter is used to express the thrombolytic enzyme. active. Although this method is intuitive, it is not very accurate for the determination of thrombolytic enzyme activity because the dissolution circle often cannot form an unconventional round shape and the measurement error is large. In addition, this method needs to consume a large amou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/37
Inventor 俞海青谢玉娟刘如林梁凤来顾晓波
Owner NANKAI UNIV