CN100529742C - Reagent kit for detecting fibrinolytic enzyme fibrinolytic activity - Google Patents
Reagent kit for detecting fibrinolytic enzyme fibrinolytic activity Download PDFInfo
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- CN100529742C CN100529742C CNB2008100172835A CN200810017283A CN100529742C CN 100529742 C CN100529742 C CN 100529742C CN B2008100172835 A CNB2008100172835 A CN B2008100172835A CN 200810017283 A CN200810017283 A CN 200810017283A CN 100529742 C CN100529742 C CN 100529742C
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Abstract
A kit for detecting fibrinolytic activity of plasmin substances is prepared by centrifugally extracting bovine blood plasma, adding cold ammonium sulfate, centrifuging and collecting precipitate; dissolving the precipitate in NaCl solution; adding cold ammonium sulfate, centrifuging and collecting precipitate; dissolving the precipitate in a PBS buffer solution with pH value of 7.4; lyophilizing to obtain bovine blood extract; and dissolving the bovine blood extract in the PBS buffer solution with pH value of 7.4, dissolving agar powder in water, mixing the two solutions, keeping the temperature at 55 DEG C for 5 min, and pouring hot mixture solution onto a flat plane to obtain the kit. The kit prepared by the method of the invention can rapidly, sensitively and accurately determine whether or not a plasmin substance has fibrinolytic activity, and has the advantages of simple operation and low detection cost in calculation of relative fibrinolytic activity as compared with standard product.
Description
Technical field
The present invention relates to a kind of detection kit, be specifically related to a kind of kit that detects fibrinolytic enzyme fibrinolytic activity.
Background technology
At present, the external detection method of relevant fibrinolytic enzyme material fibrin plate method commonly used and agarose-fibrin plate method.But said method exists the making sheet trivial operations, the reagent cost height, and measurement result is subjected to operative technique to influence shortcomings such as bigger.
Summary of the invention
The object of the present invention is to provide a kind of simple to operate, cost is low, can realize the kit and the detection method of external qualitative and quantitative analysis fibrinolytic enzyme fibrinolytic activity.
For achieving the above object, the technical solution used in the present invention is: at first get 300ml fresh bovine blood, under the rotating speed of 3500-3800r/min centrifugal 20 minutes, collect blood plasma, and add the 500ml distilled water diluting in blood plasma; Drip the 300ml saturation degree then in the blood plasma after dilution and be 80% cold ammonium sulfate, rate of addition be 2-3 drip/second, 4 ℃ leave standstill 6-8 hour after under the rotating speed at 3500-3800r/min centrifugal 15 minutes, the collecting precipitation thing; Sediment is dissolved in 150ml, in the NaCl solution of 0.1mol/L, adds the distilled water diluting of 300ml again; And then to drip the 200ml saturation degree in dilution be 80% cold ammonium sulfate, rate of addition be 2-3 drip/second, 4 ℃ leave standstill 6-8 hour after under the rotating speed at 3500-3800r/min centrifugal 15 minutes, the collecting precipitation thing; Sediment is dissolved in 150ml, 0.2mol/L, the pH value is in 7.4 the PBS damping fluid; To-60 ℃, obtained the ox blood extract in temperature-50 ℃ under the vacuum tightness 0.08Mbar in vacuum freeze drying 20-22 hour; The pH value that the ox blood extract of getting 0.5625-0.6875g is dissolved in 37.5ml is in 7.4 the PBS damping fluid, the agar powder of getting 1.000-1.125g is dissolved in and is heated to dissolving fully in the 62.5ml water, then two kinds of solution are mixed and be incorporated in 55 ℃ of insulation 5min, fall dull and stereotyped while hot and shake up, obtain detection kit.
According to the kit of method of the present invention preparation, not only can be fast, sensitivity, detect the fibrinolytic enzyme material exactly and whether possess thrombolysis activity, and can contrast with standard items, calculate relative thrombolysis activity, have simple to operately, detect advantage with low cost.
Description of drawings
Fig. 1 adopts reagent of the present invention that fibrinolytic enzyme fibrinolytic activity is measured the effect synoptic diagram;
Fig. 2 urokinase concentration and fibrinolytic area curve.
Embodiment
Embodiment 1: urokinase injection powder pin fibrinolytic is measured
Medical urokinase powder-injection is mixed with the solution of 200IU/ml.
At first get 300ml fresh bovine blood, at 3500r/min centrifugal 20 minutes, collect blood plasma, and in blood plasma, add the 500ml distilled water diluting; Drip the 300ml saturation degree then in the blood plasma after dilution and be 80% cold ammonium sulfate, rate of addition be 2-3 drip/second, 4 ℃ leave standstill 6 hours after at 3500r/min centrifugal 15 minutes, the collecting precipitation thing; Sediment is dissolved in 150ml, in the NaCl solution of 0.1mol/L, adds the distilled water diluting of 300ml again; And then to drip the 200ml saturation degree in dilution be 80% cold ammonium sulfate, rate of addition be 2-3 drip/second, 4 ℃ leave standstill 8 hours after with 3500r/min centrifugal 15 minutes, the collecting precipitation thing; Sediment is dissolved in 150ml, 0.2mol/L, the pH value is in 7.4 PBS (sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution) damping fluid; In temperature-60 ℃, vacuum freeze drying obtained the ox blood extract in 20 hours under the vacuum tightness 0.08Mbar; The pH value that the ox blood extract of getting 0.5625g is dissolved in 37.5ml is in 7.4 the PBS damping fluid, the agar powder of getting 1.000g is dissolved in the 62.5ml water, then two kinds of solution is mixed to be incorporated in 55 ℃ of insulation 5min and to obtain detectable (detectable of this dosage can produce once 7 diameters be the 9cm flat board).Detectable is poured in the flat board while hot, and rocked and make it even.After treating that flat board cools off fully, the 5ul sample is quantitatively drawn in punching, adds in the hand-hole.Flat board is added a cover, and 31 ℃ of constant temperature were hatched 8-10 hour, and promptly can be observed has tangible fibrinolytic to iris out now on flat board.When carrying out quantitative test,, can calculate relative activity by contrasting fibrinolytic circle area size with standard solution.
Embodiment 2: the fibrinolytic of Nattokinase is measured
With Nattokinase fermentation liquor suction filtration, filtrate is suitably diluted with distilled water, and is standby.
At first get 300ml fresh bovine blood, at 3700r/min centrifugal 20 minutes, collect blood plasma, and in blood plasma, add the 500ml distilled water diluting; Drip the 300ml saturation degree then in the blood plasma after dilution and be 80% cold ammonium sulfate, rate of addition be 2-3 drip/second, 4 ℃ leave standstill 7 hours after at 3700r/min centrifugal 15 minutes, the collecting precipitation thing; Sediment is dissolved in 150ml, in the NaCl solution of 0.1mol/L, adds the distilled water diluting of 300ml again; And then to drip the 200ml saturation degree in dilution be 80% cold ammonium sulfate, rate of addition be 2-3 drip/second, 4 ℃ leave standstill 6 hours after with 3700r/min centrifugal 15 minutes, the collecting precipitation thing; Sediment is dissolved in 150ml, 0.2mol/L, the pH value is in 7.4 the PBS damping fluid; In temperature-55 ℃, vacuum freeze drying obtained the ox blood extract in 20,21,22 hours under the vacuum tightness 0.08Mbar; The pH value that the ox blood extract of getting 0.6g is dissolved in 37.5ml is that the agar powder of getting 1.05g is dissolved in the 62.5ml water in 7.4 the PBS damping fluid, then two kinds of solution is mixed to be incorporated in 55 ℃ of insulation 5min and to obtain detectable, while hot detectable is fallen dull and stereotyped.After treating that flat board cools off fully, the 5ul sample is quantitatively drawn in punching, adds in the hand-hole.Flat board is added a cover, and 31 ℃ of constant temperature were hatched 8-10 hour, and promptly can be observed has tangible fibrinolytic to iris out now on flat board.When carrying out quantitative test,, can calculate relative activity by contrasting fibrinolytic circle area size with the standard urinary kinase solution.
Embodiment 3: the mensuration of urokinase concentration and fibrinolytic circle area relationship
Take by weighing urokinase standard items powder 2mg, be dissolved in the 1ml distilled water, make the standard solution of 5000IU/ml, become following concentration with distilled water diluting: 2000,1000,500,200,100 (IU/ml) is standby.
At first get 300ml fresh bovine blood, at 3800r/min centrifugal 20 minutes, collect blood plasma, and in blood plasma, add the 500ml distilled water diluting; Drip the 300ml saturation degree then in the blood plasma after dilution and be 80% cold ammonium sulfate, rate of addition be 2-3 drip/second, 4 ℃ leave standstill 8 hours after at 3800r/min centrifugal 15 minutes, the collecting precipitation thing; Sediment is dissolved in 150ml, in the NaCl solution of 0.1mol/L, adds the distilled water diluting of 300ml again; And then to drip the 200ml saturation degree in dilution be 80% cold ammonium sulfate, rate of addition be 2-3 drip/second, 4 ℃ leave standstill 7 hours after with 3800r/min centrifugal 15 minutes, the collecting precipitation thing; Sediment is dissolved in 150ml, 0.2mol/L, the pH value is in 7.4 the PBS damping fluid; In temperature-60 ℃, vacuum freeze drying obtained the ox blood extract in 22 hours under the vacuum tightness 0.08Mbar; The pH value that the ox blood extract of getting 0.6875g is dissolved in 37.5ml is in 7.4 the PBS damping fluid, the agar powder of getting 1.125g is dissolved in the 62.5ml water, then two kinds of solution are mixed being incorporated in 55 ℃ of insulation 5min and obtaining detectable, while hot detectable is poured in the flat board.After treating that flat board cools off fully, punch, quantitatively draw the standard solution of above each concentration of 5ul, put on same block of plate, 31 ℃ of constant temperature were hatched 8-10 hour, surveyed the diameter of fibrinolytic circle with vernier caliper.Calculate solusphere area and its linear relationship of mapping analysis such as Fig. 2.
Because the fibrinolytic enzyme material has the effect of good preventing and treatment thrombus.Because the principal ingredient of thrombus is a fibrin, the fibrinolytic enzyme material mainly acts on fibrin, and plays the effect of thrombolysis.Principle of the present invention is according at the external fibrinolytic that fibrinous effect and solute effect is reflected the fibrinolytic enzyme material.
Principal ingredient of the present invention is the ox blood extract, wherein mainly contains fibrinogen.By physical method, with the fibrinogen in the ox blood extract, the crosslinked fibrin that is converted into mixes with agar uniformly, is paved with check-out console in the operating process.Because the fibrinolytic enzyme material can directly act on fibrin, and with its dissolving, so near the fibrin the point sample is because dissolved, and demonstrate the edematus of agar, other parts of plank are not because there is the effect of fibrinolytic enzyme substance dissolves, and present milky (referring to Fig. 1, vertical two bigger fibrinolytic circles are live fibrinolytic effect synoptic diagram of higher fibrinolysin of enzyme, horizontal two less be the live fibrinolytic effect synoptic diagram of lower fibrinolysin of enzyme).So with the naked eye just can see very intuitively on the original milky plank, several translucent ringlets occur.Different enzymes fibrinolysin alive, to fibrinous dissolving power difference, obviously enzyme big fibrinolysin dissolving power alive is strong, and the diameter of its fibrinolytic circle is just big, and enzyme little fibrinolysin alive, the diameter of fibrinolytic circle is less.Under certain point sample amount and certain incubation conditions, the size of fibrinolytic circle and enzyme work are the better linearity relation, according to such character, the standard items that known enzyme can be lived and fibrinolytic enzyme material to be measured with time point on a plank, hatch certain hour, by detecting the diameter of fibrinolytic circle, calculate relative enzyme and live.Compared with traditional method, save and detect cost, and simple to operate.
Claims (1)
1, a kind of kit that detects fibrinolytic enzyme fibrinolytic activity is characterized in that:
1) at first gets 300ml fresh bovine blood, under the rotating speed of 3500-3800r/min centrifugal 20 minutes, collect blood plasma, and in blood plasma, add the 500ml distilled water diluting;
2) dripping the 300ml saturation degree then in the blood plasma after dilution is 80% cold ammonium sulfate, rate of addition be 2-3 drip/second, 4 ℃ leave standstill 6-8 hour after under the rotating speed at 3500-3800r/min centrifugal 15 minutes, the collecting precipitation thing;
3) sediment is dissolved in 150ml, in the NaCl solution of 0.1mol/L, adds the distilled water diluting of 300ml again;
4) drip the 200ml saturation degree and then in dilution and be 80% cold ammonium sulfate, rate of addition be 2-3 drip/second, 4 ℃ leave standstill 6-8 hour after under the rotating speed at 3500-3800r/min centrifugal 15 minutes, the collecting precipitation thing;
5) sediment is dissolved in 150ml, 0.2mol/L, the pH value is in 7.4 the PBS damping fluid;
6) in temperature-50 ℃ to-60 ℃, obtained the ox blood extract under the vacuum tightness 0.08Mbar in vacuum freeze drying 20-22 hour;
7) to be dissolved in the pH value of 37.5ml be in 7.4 the PBS damping fluid to the ox blood extract of getting 0.5625-0.6875g, the agar powder of getting 1.000-1.125g is dissolved in and is heated to dissolving fully in the 62.5ml water, then two kinds of solution are mixed and be incorporated in 55 ℃ of insulation 5min, fall dull and stereotyped while hot and shake up, obtain detection kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2008100172835A CN100529742C (en) | 2008-01-11 | 2008-01-11 | Reagent kit for detecting fibrinolytic enzyme fibrinolytic activity |
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| CNB2008100172835A CN100529742C (en) | 2008-01-11 | 2008-01-11 | Reagent kit for detecting fibrinolytic enzyme fibrinolytic activity |
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| CN101216432A CN101216432A (en) | 2008-07-09 |
| CN100529742C true CN100529742C (en) | 2009-08-19 |
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| CNB2008100172835A Expired - Fee Related CN100529742C (en) | 2008-01-11 | 2008-01-11 | Reagent kit for detecting fibrinolytic enzyme fibrinolytic activity |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019074886A1 (en) | 2017-10-09 | 2019-04-18 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
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Non-Patent Citations (4)
| Title |
|---|
| Detection of an unusually stable fibrinolytic nihibitor producedby bovine endothelial cells. D.J.Loskutoff, et al.Proc.Natl.Acad.Sci.USA,Vol.80 . 1983 |
| Detection of an unusually stable fibrinolytic nihibitor producedby bovine endothelial cells. D.J.Loskutoff, et al.Proc.Natl.Acad.Sci.USA,Vol.80 . 1983 * |
| 纳豆激酶活性测定的方法学研究. 齐香君等.中国新药杂志，第16卷第10期. 2007 |
| 纳豆激酶活性测定的方法学研究. 齐香君等.中国新药杂志，第16卷第10期. 2007 * |
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