CN103604779A - Protamine sulfate titer measuring method - Google Patents
Protamine sulfate titer measuring method Download PDFInfo
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- CN103604779A CN103604779A CN201310338351.9A CN201310338351A CN103604779A CN 103604779 A CN103604779 A CN 103604779A CN 201310338351 A CN201310338351 A CN 201310338351A CN 103604779 A CN103604779 A CN 103604779A
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Abstract
"Chinese pharmacopoeia" (2010 edition) regulates that protamine sulfate titer measurement should adopt bioassay methods. A titer of a sample is measured by comparing the clotting times of fresh rabbit bloods, pig plasmas, or rabbit plasmas which are individually added with a heparin standard substance (S) and the sample (T). The market price of rabbit plasma is high, one dose of rabbit plasma costs 10 yuan, and contains 0.5ml of rabbit plasma; and at least 60 doses are needed to measure a protamine sulfate sample. An ultraviolet spectrophotometer is adopted, the solution light-transmittance is measured under a light with a wavelength of 550 nm, then the protamine sulfate titer is measured, in the operation rabbit plasma is not needed, thus the cost is reduced, and moreover the operation is more convenient.
Description
Technical field
The present invention relates to biomedicine field, relate in particular to the mensuration that protamine sulfate is tired.
Background technology
< < Chinese Pharmacopoeia > > version appendix VII J protamine sulfate bioassay method in 2010, the assay method that protamine sulfate is tired has been described in detail in detail, by the rabbit plasma situation of condensing, has judged that it tires.Concrete operation method is as follows:
(1) it is appropriate that the preparation of heparin standard solution takes heparin standard items, adds 0.9% sodium chloride solution dissolve the solution that makes into several variable concentrations by tiring.
(2) preparation of protamine sulfate solution takes protamine sulfate, adds 0.9% sodium chloride solution, makes the solution of 1mg/ml.
(3) checking method is got 8 of small test tubes, the 1st pipe and the 8th pipe are blank pipe, add 0.9% sodium chloride solution 0.2ml, 2nd～7 pipes are test sample pipe, every pipe adds protamine sulfate solution 0.1ml, every pipe adds respectively the heparin standard items dilution 0.1ml of above-mentioned a kind of concentration again, mixes immediately.Then respectively at adding the blood plasma of 0.7ml in above-mentioned each pipe, put in 37 ± 0.5 ℃ of waters bath with thermostatic control preheating 5～10 minutes, every pipe adds respectively 1% calcium chloride solution 0.1ml, mixes immediately, avoids producing bubble, and starts the clock.Observe and record respectively and manage setting time.
Must not differ over 1.35 times the setting time of two control tube of result judgement.In the setting time of test sample pipe, be no more than in each pipe of two control tube average setting times 150%, the highest pipe of the heparin concentration of usining is as terminal pipe.Same repetition 5 times, 5 tests record the heparin concentration of terminal pipe, must not differ and are greater than 10Ge unit.The mean value of 5 results, is in protamine sulfate 1mg and the units of heparin.
The rabbit plasma using in check or in person preparation, or spend expensive price to buy, on market, the price of blood plasma is about 10 yuan/, every 0.5ml, measures a protamine sulfate sample, repeats 5 times, at least 60 rabbit plasmas, more than 600 yuan of money, inspection cost is very high.In addition, complicated operation, time-consuming is long.
Summary of the invention
Order of the present invention has been to provide a kind of protamine sulfate titration method.
Particularly, the protamine sulfate titration method the object of this invention is to provide, inapplicable rabbit plasma, checks economical, easy to operate, quick.
The present invention adopts the following assay method scheme above-mentioned purpose that is achieved:
(1) preparation of heparin standard solution takes heparin standard items, adds 0.9% sodium chloride solution and dissolves and to make into the tire heparin standard items dilution of concentration of difference.
(2) preparation of protamine sulfate solution takes protamine sulfate, adds 0.9% sodium chloride solution, is diluted to the solution of 1mg/ml.
(3) checking method is got test tube, and every pipe adds protamine sulfate solution, more every pipe adds the heparin standard items dilution of isopyknic a kind of concentration, standing, gets supernatant, under 550nm wavelength, measures the transmittance of every pipe mixed liquor.
The concentration numbers of tiring of the heparin standard items of transmittance maximum tube, is tiring of protamine sulfate.
Compared with prior art, advantage of the present invention and good effect:
Through many experiments, tiring of the protamine sulfate that the present invention measures, with the protamine sulfate bioassay method of < < Chinese Pharmacopoeia > > version regulation in 2010 measured tire in full accord.But contrast discovery, advantage of the present invention is that the experiment reagent 1. using is few, does not need rabbit plasma, lime chloride, and experimental cost is low; 2. swift to operate, the time is short; 3. test required instrument few, do not need water-bath, stopwatch etc.
Embodiment
Embodiment 1:
(1) preparation of heparin standard solution
Take heparin standard items 150.05mg, the 180iu/mg that tires of unit, adds 0.9% sodium chloride solution and dissolves and make it to become tire each 25ml of heparin standard items dilution of concentration of difference, the concentration of tiring is respectively 145iu/ml, 150iu/ml, 155iu/ml, 160iu/ml, 165iu/ml, 170iu/ml.
(2) preparation of protamine sulfate solution
Take protamine sulfate 122.52mg, lot number 110901, adds 0.9% sodium chloride solution, is diluted to the solution of 1mg/ml.
(3) checking method
Get 6, test tube, in each test tube, add protamine sulfate solution 5ml, then in each test tube, to add respectively the corresponding concentration of 5ml be 145iu/ml, 150iu/ml, 155iu/ml, 160iu/ml, 165iu/ml, 170iu/ml heparin standard items dilution, standing, get supernatant, under 500nm wavelength, measure the transmittance of every pipe mixed liquor, repeat 3 times.
The results are shown in following table:
By transmittance, detect, the transmittance of the concentration 155iu/ml pipe of tiring of heparin standard items is the highest, is tiring as 155iu/mg of protamine sulfate.
(4) existing checking method
Get 8, test tube, the 1st pipe and the 8th pipe, for blank pipe, add 0.9% sodium chloride solution 0.2ml, 2nd～7 pipes are test sample pipe, every pipe adds protamine sulfate solution 0.1ml, more every pipe adds respectively the heparin standard items dilution 0.1ml of above-mentioned a kind of concentration, mixes immediately.Then respectively at adding the blood plasma of 0.7ml in above-mentioned each pipe, put in 37 ± 0.5 ℃ of waters bath with thermostatic control preheating 5～10 minutes, every pipe adds respectively 1% calcium chloride solution 0.1ml, mixes immediately, avoids producing bubble, and starts the clock.Observe and record respectively and manage setting time.
Embodiment 2:
(1) preparation of heparin standard solution
Take heparin standard items 152.35mg, the 180iu/mg that tires of unit, adds 0.9% sodium chloride solution and dissolves and make it to become tire each 25ml of heparin standard items dilution of concentration of difference, concentration is respectively 145iu/ml, 150iu/ml, 155iu/ml, 160iu/ml, 165iu/ml, 170i u/ml.
(2) preparation of protamine sulfate solution
Take protamine sulfate 125.30mg, lot number 110902, adds 0.9% sodium chloride solution, is diluted to the solution of 1mg/ml.
(3) checking method
Get 6, test tube, in each test tube, add protamine sulfate solution 5ml, then in each test tube, to add respectively the corresponding concentration of 5ml be 145iu/ml, 150iu/ml, 155iu/ml, 160i u/ml, 165iu/ml, 170iu/ml heparin standard items dilution, standing, get supernatant, under 450nm wavelength, measure the transmittance of mixed liquor in each test tube, repeat 3 times.
The results are shown in following table:
By transmittance, detect, the transmittance of the concentration 150iu/ml pipe of tiring of heparin standard items is the highest, is tiring as 150iu/mg of protamine sulfate.
(4) existing checking method
Get 8, test tube, the 1st pipe and the 8th pipe, for blank pipe, add 0.9% sodium chloride solution 0.2ml, 2nd～7 pipes are test sample pipe, every pipe adds protamine sulfate solution 0.1ml, more every pipe adds respectively the heparin standard items dilution 0.1ml of above-mentioned a kind of concentration, mixes immediately.Then respectively at adding the blood plasma of 0.7ml in above-mentioned each pipe, put in 37 ± 0.5 ℃ of waters bath with thermostatic control preheating 5～10 minutes, every pipe adds respectively 1% calcium chloride solution 0.1ml, mixes immediately, avoids producing bubble, and starts the clock.Observe and record respectively and manage setting time.
From embodiment 1,2, can find out, adopt the protamine sulfate of the present invention method of inspection of tiring, simple and easy to do, required testing reagent is few, and cost is low, and valence value is measured accurately same.
The above, be only preferred embodiment of the present invention, is not the present invention to be done to the restriction of other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not depart from, any simple modification, equivalent variations and the remodeling above embodiment done according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.
Claims (9)
1. the assay method that protamine sulfate is tired, is characterized in that comprising the sodium chloride solution that uses heparin.
2. assay method claimed in claim 1, is characterized in that comprising the following steps:
(1) preparation of heparin standard solution
Take heparin standard items, add sodium chloride solution and dissolve and to make it to become the tire heparin standard items dilution of concentration (iu/ml) of difference.
(2) preparation of protamine sulfate solution
Take protamine sulfate testing sample, add sodium chloride solution, be diluted to protamine sulfate solution.
(3) checking method is got test tube, in every test tube, add respectively resulting protamine sulfate solution in step (2), in every test tube, add respectively again the tire heparin standard items dilution of concentration of the difference of preparation in above-mentioned steps (1), standing, get supernatant, measure the transmittance of mixed liquor in each test tube, wherein the protamine sulfate solution in each test tube equates with the volume of heparin standard items dilution.
According to the corresponding valence value of the concentration numbers of tiring of the heparin standard items dilution in the highest test tube of transmittance, the unit that calculates protamine sulfate sample tire (iu/mg).
3. assay method claimed in claim 2, is characterized in that: in described step (1) and (2), the concentration of sodium chloride solution used is 0.8 quality %-1.2 quality %.
4. assay method claimed in claim 2, is characterized in that: the concentration of the protamine sulfate solution in described step (2) is 0.6-1.2mg/ml.
5. assay method claimed in claim 4, is characterized in that: the concentration of the protamine sulfate solution in described step (2) is 1mg/ml.
6. assay method claimed in claim 2, is characterized in that: the concentration difference of tiring that the difference in described step (1) is tired between the heparin standard items dilution of concentration can be identical or different.
7. assay method claimed in claim 2, is characterized in that: the concentration difference of tiring that the difference in described step (1) is tired between the heparin standard items dilution of concentration is 3-8iu/ml.
8. assay method claimed in claim 2, is characterized in that: the light wavelength of measuring transmittance in described step (3) is 400nm-600nm.
9. assay method claimed in claim 2, is characterized in that: in described step (1), the tire concentration range of tiring of heparin standard items dilution of concentration of difference is 100iu/m1-200iu/ml.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3915640A (en) * | 1974-08-06 | 1975-10-28 | Warner Lambert Co | Method and composition for detecting fibrin monomers and fibrin degradation products |
| US4175182A (en) * | 1978-07-03 | 1979-11-20 | Research Corporation | Separation of high-activity heparin by affinity chromatography on supported protamine |
| US4551308A (en) * | 1981-07-09 | 1985-11-05 | International Technidyne Corp. | Apparatus for analyzing the influence of additive reagents upon the coagulation of blood and related methods |
| WO1998038263A1 (en) * | 1997-02-27 | 1998-09-03 | St. John's University | Optical membrane films for polycation detection |
| US20050023153A1 (en) * | 2003-07-09 | 2005-02-03 | Auburn University | Reversible electrochemical sensors for polyions |
| CN101357935A (en) * | 2008-09-03 | 2009-02-04 | 河北科技大学 | Method for separating purified protamine using reverse micelle method |
| CN102445483A (en) * | 2011-09-30 | 2012-05-09 | 中国科学院烟台海岸带研究所 | Method for detecting heparins |
-
2013
- 2013-07-30 CN CN201310338351.9A patent/CN103604779B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3915640A (en) * | 1974-08-06 | 1975-10-28 | Warner Lambert Co | Method and composition for detecting fibrin monomers and fibrin degradation products |
| US4175182A (en) * | 1978-07-03 | 1979-11-20 | Research Corporation | Separation of high-activity heparin by affinity chromatography on supported protamine |
| US4551308A (en) * | 1981-07-09 | 1985-11-05 | International Technidyne Corp. | Apparatus for analyzing the influence of additive reagents upon the coagulation of blood and related methods |
| WO1998038263A1 (en) * | 1997-02-27 | 1998-09-03 | St. John's University | Optical membrane films for polycation detection |
| US20050023153A1 (en) * | 2003-07-09 | 2005-02-03 | Auburn University | Reversible electrochemical sensors for polyions |
| CN101357935A (en) * | 2008-09-03 | 2009-02-04 | 河北科技大学 | Method for separating purified protamine using reverse micelle method |
| CN102445483A (en) * | 2011-09-30 | 2012-05-09 | 中国科学院烟台海岸带研究所 | Method for detecting heparins |
Non-Patent Citations (2)
| Title |
|---|
| BRITISH PHARMACOPOEIA COMMISSION: "《British Pharmacopoeia VolumeⅠ&Ⅱ》", 31 December 2012, article "Ptotamine Sulfate" * |
| 王巍 等: "《中国药典2010年版第2部》", 31 December 2010, article "附录Ⅻ J 硫酸鱼精蛋白生物测定法" * |
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