CN1763219A - Fibrinogen plate assay for detection of thrombin, thrombin-like enzyme and thrombin inhibitor - Google Patents
Fibrinogen plate assay for detection of thrombin, thrombin-like enzyme and thrombin inhibitor Download PDFInfo
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- CN1763219A CN1763219A CN 200510019588 CN200510019588A CN1763219A CN 1763219 A CN1763219 A CN 1763219A CN 200510019588 CN200510019588 CN 200510019588 CN 200510019588 A CN200510019588 A CN 200510019588A CN 1763219 A CN1763219 A CN 1763219A
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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
The invention belongs to bio protease particularly zymoplasm, Thrombin-like enzyme and thrombin inhibitors detection method field.
Background technology
Zymoplasm, Thrombin-like enzyme and thrombin inhibitors (as r-hirudin, heparin) etc. extensively are present in the animal and plant, are the main research objects of medical science, physiotechnology and bio-pharmaceuticals.It is scientific research and industrial essential condition that zymoplasm, Thrombin-like enzyme and inhibitor thereof are carried out biological activity determination.The biological activity assay method of having set up at present mainly contains two kinds: zymoplasm volumetry and zymoplasm chromogenic substrate method.The zymoplasm volumetry is comparatively easy, quick because of its operation, is most widely used.But have that the difference with the naked eye judge zero pour (being reaction end) is big, the turbidity of inspection product or the mutual interference of color phase, susceptibility low (detecting every milliliter 10 enzyme activity unit), problem such as quantitative inaccurate.Behind the chromogenic substrate that synthetic has been arranged, realized the detection by quantitative of zymoplasm with zymoplasm chromogenic substrate method, its susceptibility greatly improves (detecting every milliliter 2.5 enzyme activity unit); But because chromogenic substrate reagent is too expensive, appointed condition is had relatively high expectations during mensuration, and inspection product turbidity or color also are subject to disturb, and its application is restricted.These further investigation and development and use to zymoplasm, thrombin inhibitors, Thrombin-like enzyme aspects such as (snake venom fiber eliminating enzymes) bring certain difficulty.
Summary of the invention
The technical problem to be solved in the present invention provides bioactive easy, stable, the high sensitive of a kind of detection by quantitative zymoplasm, Thrombin-like enzyme and thrombin inhibitors, detection method cheaply.
The present invention solves the problems of the technologies described above with following technical scheme:
Adopt fibrinogen plate assay detection by quantitative zymoplasm, Thrombin-like enzyme and thrombin inhibitors biological activity, method is:
One, making sheet: will heat abundant dissolved agarose and with 0.1M pH7.4 phosphoric acid buffer dissolved Fibrinogen, be 1: 1 mixed with the weight ratio volume, irritates plate in plate, promptly makes the Fibrinogen agar plate after the cooling.
Two, punching: after the cooling of Fibrinogen agar plate, punching at a certain distance.
Three, the preparation of standard substance, inspection product and application of sample reaction:, and be diluted to series concentration with 0.05M pH7.4 phosphoric acid buffer dissolving standard substance.With same damping fluid dissolving inspection product and suitably dilution of do.With the standard substance after the dilution, inspection product application of sample in above-mentioned plate hole respectively.Plate was added a cover rearmounted 37 ℃ of constant temperature diffusion reactions 7-10 hour.
Four, measure reacting hole precipitation circle, the drawing standard curve, calculation result: behind Fibrinogen agar plate application of sample diffusion reaction, but naked eyes see that well forms the oyster white circle from inside to outside and precipitates circle, and the size of precipitation circle becomes positive correlation with enzyme activity concentration.Measure each standard substance reacting hole precipitation loop diameter, on semi-logarithmic coordinate paper, draw the enzyme activity unit typical curve.Measure each inspection product reacting hole precipitation loop diameter, can find its enzyme activity unit number on typical curve, the extension rate that multiply by the inspection product is the actual enzyme activity unit number of inspection product.
The fibrinogen plate assay of detection zymoplasm of the present invention, Thrombin-like enzyme and thrombin inhibitors, easy and simple to handle, directly perceived, stable, susceptibility is high (detecting every milliliter 0.15 enzyme activity unit), be not subjected to inspection product turbidity or color interference, with low cost.As long as in common lab, there is thermostat container to implement, be a kind of experimental technique that is easy to promote the use of.
Description of drawings
Fig. 1 carries out the photo that Thrombin-like enzyme detects the Fibrinogen flat board of usefulness.
Fig. 2 is the canonical plotting of embodiment 1.
Fig. 3 is the canonical plotting of embodiment 2.
Fig. 4 is the canonical plotting of embodiment 3.
Fig. 5 is the canonical plotting of embodiment 4.
Embodiment
The present invention utilizes Fibrinogen to be substrate, use agarose and Fibrinogen to be made into the Fibrinogen agar plate, the diffusion reaction on this flat board by standard substance and inspection product carries out detection by quantitative to zymoplasm, Thrombin-like enzyme and thrombin inhibitors.Its ultimate principle is to be fixed in the substrate-Fibrinogen of enzyme on the agar plate equably, carry out diffusion reaction by in the sample hole, adding enzyme solution, enzyme in diffusion process with substrate generation hydrolysis reaction, make Fibrinogen be hydrolyzed into scleroproein, and the crosslinked precipitation that is creamy white take place.White precipitate circle size is proportionate with enzyme activity.Zymoplasm, the Thrombin-like enzyme standard substance of known unit of activity are diluted to series concentration and sample to be checked suitably dilutes, carry out diffusion reaction, can draw out corresponding enzyme activity typical curve by measuring reacting hole precipitation circle size at the Fibrinogen flat board.Measure simultaneously that inspection product reacting hole precipitation circle is big or small under the same conditions, can on typical curve, find respective value and reach the purpose of detection by quantitative.
The key step that detects zymoplasm and Thrombin-like enzyme is:
One, make the Fibrinogen agar plate:
(1) preparation weight percent (as follows) is 2.5% agarose, and heating is dissolving fully.
(2) Fibrinogen dissolves with 0.1M pH7.4 phosphoric acid buffer, with above-mentioned agarose (being heated to 90～100 ℃) be 1: 1 mixed with the weight ratio volume, in plate, irritate plate, irritate plate thickness and be about 2 millimeters, promptly make the Fibrinogen agar plate after the cooling.
Two, punching: on the Fibrinogen agar plate of cooled and solidified, by 1.6 centimetres spacing punching.The aperture is about 4 millimeters.
Three, the preparation of standard substance, inspection product and application of sample reaction:, and be diluted to series concentration with 0.05M pH7.4 phosphoric acid buffer dissolving standard substance.Require to do suitably dilution with same damping fluid dissolving inspection product and according to difference.With the standard substance after the dilution, inspection product application of sample in above-mentioned Fibrinogen agar plate plate hole respectively, every hole application of sample 18 μ l.Plate was added a cover rearmounted 37 ℃ of constant temperature diffusion reactions 7-10 hour.
Four, drawing standard curve, calculation result: behind Fibrinogen agar plate application of sample diffusion reaction, measure each standard substance reacting hole precipitation loop diameter (millimeter), on semi-logarithmic scale paper, draw the enzyme activity typical curve.Measure each inspection product reacting hole precipitation loop diameter, can find its corresponding enzyme activity unit on typical curve, the extension rate that multiply by the inspection product is the actual unit of enzyme activity number of inspection product.
The active detection by quantitative step of thrombin inhibitors-r-hirudin is:
R-hirudin inspection product with the dissolving of 0.05M pH7.4 phosphoric acid buffer, are done 2 times, 4 times, 8 times, 16 times dilutions.Mix with the r-hirudin of above-mentioned dilution respectively with 1: 1 ratio with 2.5U/ml zymoplasm standard substance that (extension rate that mixes the back r-hirudin this moment should be 4 times, 8 times, 16 times, 32 times, concentration of thrombin should be 1.25U/ml), put 37 ℃ of reactions 5 minutes, can on the Fibrinogen agar plate, distinguish application of sample with the zymoplasm standard substance of each weaker concn, every hole application of sample 18 μ l, plate added a cover rearmounted 37 ℃ of constant temperature diffusion reactions 7-10 hour.According to diffusion reaction production standard curve, measure r-hirudin inspection product hole, can on typical curve, find the residue units of each hole zymoplasm.The biological activity of r-hirudin is to represent an antithrombin unit of r-hirudin (ATU) with the amount of the zymoplasm that suppresses an activity unit.Amount with the zymoplasm (1.25U/ml) of participating in reaction is subtracted each other with the residual content of reaction back zymoplasm, is multiplied by the leech extension rate again, can calculate the substantial activity unit of r-hirudin inspection product.
Embodiment 1: the detection by quantitative of zymoplasm
1. prepare 2.5% agarose: take by weighing 2.5 gram agaroses, put in the flask, add distilled water to 100ml, put about 30 minutes of 100 ℃ of water-baths, it is standby that agarose is fully dissolved.
2. now join 0.1% (percentage concentration, as follows) Fibrinogen damping fluid: take by weighing human fibrinogen 8mg, with 0.1M pH7.4 phosphoric acid buffer 8ml dissolving.
3. making sheet: cut-off directly is 9 centimetres a plate, on the table top of placement level (measuring with level bar).2.5% agarose for preparing is heated to 90～100 ℃, gets 8ml and 0.1% Fibrinogen damping fluid 8ml mixing, pour into immediately in the plate, be the Fibrinogen flat board after the room temperature cooling.
4. punching: on cooled Fibrinogen agar plate, by 1.6 centimetres spacing punching.The aperture is 4 millimeters.
5. the preparation of standard substance and inspection product and application of sample reaction:, and be diluted to 5.0U/ml, 2.5U/ml, 1.25U/ml, 0.62U/ml, 0.31U/ml, 0.15U/ml series concentration with 0.05M pH7.4 phosphoric acid buffer dissolving standard substance.With same damping fluid 1ml dissolving inspection product, and do 20 times, 40 times, 80 times dilutions.With the standard substance after the dilution, inspection product application of sample in above-mentioned plate hole respectively, every hole application of sample 18 μ l.Plate was added a cover rearmounted 37 ℃ of constant temperature diffusion reactions 9 hours.
6. drawing standard curve, calculation result: use each reacting hole precipitation loop diameter (mm) size of oil ga(u)ge kind of calliper; Standard orifice: by the order of 5 → 0.15U/ml, the precipitation loop diameter is followed successively by 11.12mm, 10.06mm, 9.22mm, 8.26mm, 7.30mm, 6.36mm;
Inspection product hole: get and drop on typical curve intermediary inspection product hole and calculate, diluting 80 times is 8.98mm, and the enzymic activity of finding on following typical curve is 1.07U/ml, is multiplied by the extension rate 80 of the product of inspection, and the actual enzymic activity of examining product is 85.6 units/ml.
Embodiment 2: the detection by quantitative of zymoplasm
Step is 1.～4. with embodiment 1.
5. the preparation of standard substance and inspection product and application of sample reaction:, and be diluted to 5.0U/ml, 2.5U/ml, 1.25U/ml, 0.62U/ml, 0.31U/ml, 0.15U/ml series concentration with 0.05M pH7.4 phosphoric acid buffer dissolving standard substance.With same damping fluid 1ml dissolving inspection product, and do 160 times of dilutions.With the standard substance after the dilution, inspection product application of sample in above-mentioned plate hole respectively, every hole application of sample 18 μ l.Plate was added a cover rearmounted 37 ℃ of constant temperature diffusion reactions 7 hours.
6. drawing standard curve, calculation result: use each reacting hole precipitation loop diameter size of oil ga(u)ge kind of calliper, standard orifice: by the order of 5 → 0.15U/ml, the precipitation loop diameter is followed successively by 7.94mm, 7.62mm, 7.20mm, 6.78mm, 6.24mm, 5.94mm;
Inspection product hole: diluting 160 times is 6.36mm, and the enzymic activity that detects on following typical curve is 0.32U/ml, is multiplied by the extension rate 160 of inspection product, and the actual enzymic activity of inspection product is 51.2U/ml.
Embodiment 3: the detection by quantitative of Thrombin-like enzyme
1. with embodiment 1.
2. now join 0.15% Fibrinogen damping fluid: take by weighing human fibrinogen 12mg, with 0.1M pH7.4 phosphoric acid buffer 8ml dissolving.
3. making sheet: cut-off directly is 9 centimetres a plate, on the table top of placement level (measuring with level bar).2.5% agarose for preparing is heated to 90～100 ℃, gets 8ml and 0.15% Fibrinogen damping fluid 8ml mixing, pour into immediately in the plate, be the Fibrinogen flat board after the room temperature cooling.
4. punching: with embodiment 1.
5. the preparation of standard substance and inspection product and application of sample reaction:, and be diluted to 1.25U/ml, 0.62U/ml, 0.31U/ml, 0.15U/ml, 0.075U/ml series concentration with 0.05M PH7.4 phosphoric acid buffer dissolving standard substance.With same damping fluid 1ml dissolving inspection product, and do 10 times of dilutions.With the standard substance after the dilution, inspection product application of sample in above-mentioned plate hole respectively, every hole application of sample 18 μ l.Plate was added a cover rearmounted 37 ℃ of constant temperature diffusion reactions 10 hours.
6. drawing standard curve, calculation result: use each reacting hole precipitation loop diameter size of oil ga(u)ge kind of calliper; Standard orifice: by the order of 1.25 → 0.075U/ml, the precipitation loop diameter is followed successively by 12.28mm, 10.9mm, 9.52mm, 8.04mm, 6.56mm;
Inspection product hole: diluting 10 times is 10.68mm, and the enzymic activity that detects on following typical curve is 0.56U/ml, is multiplied by the extension rate 10 of inspection product, and the actual enzymic activity of inspection product is 5.6U/ml.
The bioactive detection by quantitative of embodiment 4 thrombin inhibitorss
Making sheet, the punching with embodiment 1 1.～4.;
Standard substance and the inspection preparation of product and application of sample reaction: zymoplasm standard substance series concentration dilution with embodiment 1 5., r-hirudin inspection product use 0.05M pH7.4 phosphoric acid buffer 1ml to dissolve, and do 2 times, 4 times, 8 times, 16 times dilutions.Mixes with the r-hirudin of above-mentioned dilution respectively by 1: 1 ratio with the good 2.5U/ml zymoplasm standard substance of dilution that (extension rate of r-hirudin should be 4 times, 8 times, 16 times, 32 times after mixing this moment, concentration of thrombin should be 1.25U/ml), put 37 ℃ of reactions 5 minutes, can be on the Fibrinogen agar plate and standard substance application of sample simultaneously, every hole application of sample 18 μ l, plate added a cover rearmounted 37 ℃ of constant temperature diffusion reactions 8 hours.
The drawing standard curve, calculation result: use each reacting hole precipitation loop diameter size of oil ga(u)ge kind of calliper;
Standard orifice: by the order of 5 → 0.15U/ml, the precipitation loop diameter is followed successively by 10.88mm, 9.74mm, 8.88mm, 7.8mm, 6.90mm, 6.48mm;
Inspection product hole: get and drop on the typical curve intermediary and examine the product hole and calculate, diluting 16 times is 8.18mm, the residue units of finding zymoplasm on following typical curve is 0.7U/ml, and the amount of the zymoplasm that is suppressed by r-hirudin should be: 1.25U/ml-0.7U/ml=0.55ATU/ml.Be multiplied by the leech extension rate again, the reality of r-hirudin inspection product is 8.8ATU/ml.
Claims (2)
1. a Fibrinogen plate method that detects zymoplasm, Thrombin-like enzyme and thrombin inhibitors utilizes Fibrinogen to be substrate, it is characterized in that
Step is:
A. making sheet: will heat abundant dissolved agarose and with 0.1M pH7.4 phosphoric acid buffer dissolved Fibrinogen, be 1: 1 mixed with the weight ratio volume, irritates plate in plate, promptly makes the Fibrinogen agar plate after the cooling;
B. punching: after the cooling of Fibrinogen agar plate, punching at a certain distance;
C. the preparation of standard substance, inspection product and application of sample reaction:, and be diluted to series concentration with 0.05M pH7.4 phosphoric acid buffer dissolving standard substance; With same damping fluid dissolving inspection product and suitably dilution of do; With the standard substance after the dilution, inspection product application of sample in above-mentioned plate hole respectively.Plate was added a cover rearmounted 37 ℃ of constant temperature diffusion reactions 7-10 hour;
D. measure reacting hole precipitation circle, the drawing standard curve, calculation result: behind Fibrinogen agar plate application of sample diffusion reaction, but naked eyes see that well forms the circular precipitation of oyster white from inside to outside and encloses, and the size of precipitation circle becomes positive correlation with enzyme activity concentration; Measure each standard substance reacting hole precipitation loop diameter, on semi-logarithmic coordinate paper, draw the enzyme activity unit typical curve; Measure each inspection product reacting hole precipitation loop diameter, can find its enzyme activity unit number on typical curve, the extension rate that multiply by the inspection product is the actual enzyme activity unit number of inspection product.
2. as the Fibrinogen plate method of the described detection zymoplasm of claims, Thrombin-like enzyme and thrombin inhibitors, the active detection by quantitative step that it is characterized in that thrombin inhibitors-r-hirudin is: r-hirudin inspection product are dissolved with 0.05M pH7.4 phosphoric acid buffer, and make suitable multiple and dilute, the zymoplasm standard substance with 1: 1 ratio respectively with above-mentioned dilution after the r-hirudin of different concns mix postpone slightly, promptly on the Fibrinogen agar plate, distinguish application of sample, constant temperature diffusion reaction with the zymoplasm standard substance of each weaker concn; According to diffusion reaction production standard curve, measure r-hirudin inspection product hole, can on typical curve, find the residue units of each hole zymoplasm; Amount with the zymoplasm of participating in reaction is subtracted each other with the residual content of reaction back zymoplasm, is multiplied by the leech extension rate again, can calculate the substantial activity unit of r-hirudin inspection product.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101680905A (en) * | 2008-05-22 | 2010-03-24 | 伊西康公司 | Protein analysis |
| CN103376329A (en) * | 2012-04-19 | 2013-10-30 | 蓬莱诺康药业有限公司 | Characterization method of stypticity of hemocoagulase atrox for injection and effective components thereof |
| CN104862374A (en) * | 2015-05-12 | 2015-08-26 | 浙江大学舟山海洋研究中心 | Method for detecting collagen enzymolysis activity of trepang cathepsin |
| CN104965017A (en) * | 2015-06-17 | 2015-10-07 | 广西师范学院 | Method of determining anti-thrombin activity of hirudin |
| CN113337575A (en) * | 2021-07-29 | 2021-09-03 | 湖北真福医药有限公司 | Detection method for nattokinase activity |
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2005
- 2005-10-11 CN CN 200510019588 patent/CN1763219A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101680905A (en) * | 2008-05-22 | 2010-03-24 | 伊西康公司 | Protein analysis |
| CN101680905B (en) * | 2008-05-22 | 2013-11-27 | 伊西康公司 | Protein assay |
| US9213035B2 (en) | 2008-05-22 | 2015-12-15 | Ethicon, Inc. | Protein assay |
| US9896716B2 (en) | 2008-05-22 | 2018-02-20 | Ethicon, Inc. | Protein assay |
| CN103376329A (en) * | 2012-04-19 | 2013-10-30 | 蓬莱诺康药业有限公司 | Characterization method of stypticity of hemocoagulase atrox for injection and effective components thereof |
| CN104862374A (en) * | 2015-05-12 | 2015-08-26 | 浙江大学舟山海洋研究中心 | Method for detecting collagen enzymolysis activity of trepang cathepsin |
| CN104965017A (en) * | 2015-06-17 | 2015-10-07 | 广西师范学院 | Method of determining anti-thrombin activity of hirudin |
| CN104965017B (en) * | 2015-06-17 | 2017-06-16 | 广西师范学院 | The assay method of hirudin anticoagulant hemase activity |
| CN113337575A (en) * | 2021-07-29 | 2021-09-03 | 湖北真福医药有限公司 | Detection method for nattokinase activity |
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