CN106872463A - Dynamic turbidimetric is used for endotoxic quantitative detecting method in alginate material - Google Patents
Dynamic turbidimetric is used for endotoxic quantitative detecting method in alginate material Download PDFInfo
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- CN106872463A CN106872463A CN201510920871.XA CN201510920871A CN106872463A CN 106872463 A CN106872463 A CN 106872463A CN 201510920871 A CN201510920871 A CN 201510920871A CN 106872463 A CN106872463 A CN 106872463A
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- endotoxin
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- alginate
- alginate material
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Abstract
Dynamic turbidimetric is used for endotoxic quantitative determination in alginate material the invention discloses one kind, the method can provide endotoxic absolute value in alginate material.
Description
Technical field
It is specifically a kind of by dynamic turbidimetric the present invention relates to a kind of method for quantitatively determining of endotoxin content
For endotoxic quantitative determination in alginate material.
Background technology
Alginate material shows extensively because of its good biocompatibility as the biomaterial of regeneration
Wealthy application prospect.But endotoxin will have a strong impact on its biofacies in application process in alginate material
Capacitive.Used as the raw material product of organizational project, the endotoxin composition in its product will be strictly limited.Mesh
Before, for endotoxic detection method with gel limitation method in relevant national standard.The shortcoming of the method is
Because sensitivity of the limulus reagent is poor, it is impossible to meet high-level (implantation level or organizational project level) alginate material
The quantitatively characterizing of endotoxin content absolute value in product.
The content of the invention
Regarding to the issue above, present invention proposition, determines senior by high sensitivity TAL by dynamic turbidimetric
Endotoxic quantitative determination in other alginate material.
Concrete technical scheme proposed by the present invention is as follows：
(1) utensil needed to use in operating process carries out endotoxin-free pretreatment.
(2) with tiny electrolytic cell water dissolves endotoxin standard, it is placed in more than suspension device suspension 15min.
(3) with tiny electrolytic cell dilute with water endotoxin standard solution in the range of 2-0.03EU/mL,
Every 2 times are diluted to a test point.During dilution, mixing time is 30s.
(4) with tiny electrolytic cell water dissolves high sensitivity TAL, note not producing bubble as far as possible.
(5) the μ L of each concentration endotoxin 100 are taken in detection pipe, plus the μ L of TAL 100, suspension device
Instrument for testing endotoxin detection (37 DEG C of detection temperature, Detection wavelength 660nm), endotoxin inspection are placed in after gently mixing
It is negative control to look into water.When the reaction time of the reaction time more than standard curve least concentration of negative control,
Total data is carried out into linear regression analysis.According to linear regression analysis, the coefficient correlation (r) of standard curve
Absolute value should be greater than or equal to 0.980, test as effective.
(6) alginate solution is configured：Tiny electrolytic cell prepares alginate solution, solution concentration with water
It is 20 μ g/ml-20mg/ml.The endotoxin solution for preparing concentration known is used for interference test, determines and reclaims
Rate.
(7) the μ L of step (6) alginate sample solution 100 are taken in detection pipe, 100 μ L are added
TAL prepared by step (4), it is light mixed after instrument for testing endotoxin detection (37 DEG C of detection temperature, detection
Wavelength 660nm).The standard curve drawn with step (5) demarcates endotoxin content in testing sample.When interior
The rate of recovery of toxin is between 50%~200%, then it is assumed that the measured value under this experimental condition is effective.
The quantitative detecting method of protein content in measure alginate material proposed by the present invention, is adapted to determine and divides
Alginate material of the son amount in 10-500kDa.
The quantitative detecting method of protein content in measure alginate material proposed by the present invention, is adapted to determine sea
The sodium salt or sylvite of alginic acid.
≤ 0.03EU/mL is wanted in the sensitivity of high sensitivity TAL used in the present invention.
The endotoxin content of tiny electrolytic cell water used in the present invention is less than 0.005EU/mL.
Specific embodiment
(1) with tiny electrolytic cell water dissolves endotoxin standard, it is placed in more than suspension device suspension 15min.
(2) with tiny electrolytic cell dilute with water endotoxin standard solution in the range of 2-0.03EU/mL,
Every 2 times are diluted to a test point.During dilution, mixing time is 30s.Specific concentration gradient and volume are such as
Under：
2EU/mL 1mL, 1EU/mL 5mL, 0.5EU/mL 2mL, 0.25EU/mL 1mL,
0.125EU/mL 1mL, 0.0625EU/mL 1mL, 0.03125EU/mL 1mL.
(3) with tiny electrolytic cell water dissolves high sensitivity TAL (sensitivity of the limulus reagent 0.03EU/mL),
Note not producing bubble as far as possible.
(4) each concentration endotoxin standard solution that step (2) is prepared is taken into 100 μ L in detection pipe,
Plus the μ L of TAL 100, suspension device is placed in instrument for testing endotoxin detection after gently mixing (37 DEG C of detection temperature is examined
Survey wavelength 660nm), tiny electrolytic cell water is negative control.When the reaction time of negative control is more than mark
In the reaction time of directrix curve least concentration, total data is carried out into linear regression analysis.According to linear regression point
Analysis, the absolute value of the coefficient correlation (r) of standard curve is equal to 0.992.
(5) sodium alginate sample solution is configured：Tiny electrolytic cell prepares sodium alginate sample solution with water,
Solution concentration is 1mg/ml.
(6) the μ L of alginate sample solution 100 of step (5) preparation are taken in detection pipe, is added
TAL prepared by 100 μ L steps (3) is gently mixed, while taking the 1EU/mL endogenous toxic materials of step (2) preparation
In detection pipe, the TAL for adding 100 μ L steps (3) to prepare gently is mixed the μ L of plain standard solution 100,
Above-mentioned two groups of mixed liquors are detected into (37 DEG C of detection temperature, Detection wavelength 660nm) in instrument for testing endotoxin,
Endotoxin content is 500EU/g in obtaining testing sample according to standard curve.1EU/mL endotoxin standards
The endotoxic rate of recovery that sample sets are determined is 95%, it was demonstrated that the measured value under this experimental condition is effective.
Claims (8)
1. dynamic turbidimetric is used for endotoxic quantitative detecting method, its feature in alginate material
It is：TAL is used for endotoxic quantitative determination in alginate material, measurement result can provide sea
Endotoxic absolute value in alginate material.
2. according to the quantitative determination for determining endotoxin content in alginate material described in claim 1
Method, it is characterised in that methods described is comprised the following steps that：
(1) with tiny electrolytic cell water dissolves endotoxin standard, be placed in suspension device suspension 15min with
On；
(3) with tiny electrolytic cell dilute with water endotoxin standard solution in the range of 2-0.03EU/mL,
Every 2 times are diluted to a test point, i.e., what each endotoxin concns halved successively is diluted to a detection sample
Product liquid；During dilution, mixing time is more than 30s；
(4) with tiny electrolytic cell water dissolves TAL；
(5) the μ L of each concentration endotoxin 100 are taken in detection pipe, plus the μ L of TAL 100, suspension device
Instrument for testing endotoxin is placed in after gently mixing and detects the reaction time (37 DEG C of detection temperature, Detection wavelength 660nm),
Tiny electrolytic cell water is negative control；It is minimum dense more than in standard curve when the reaction time of negative control
During the reaction time of the standard items of degree, total data is carried out into linear regression analysis；
(6) alginate solution is configured：Tiny electrolytic cell prepares alginate solution with water, and solution is dense
It is 20 μ g/ml-20mg/ml to spend；
(7) the μ L of step (6) alginate sample solution 100 are taken in detection pipe, 100 μ L are added
TAL prepared by step (4), mixes after instrument for testing endotoxin detection (37 DEG C of detection temperature, detection
Wavelength 660nm)；The standard curve drawn with step (5) demarcates endotoxin content in testing sample.
3. quantified according to endotoxin content in the measure alginate material described in claim 1 or 2
Detection method, it is characterised in that：The method is adapted to determine alginate material of the molecular weight in 10-500kDa
Material.
4. quantified according to endotoxin content in the measure alginate material described in claim 1 or 2
Detection method, it is characterised in that：The method is adapted to determine the sodium salt or sylvite of alginic acid.
5. quantified according to endotoxin content in the measure alginate material described in claim 1 or 2
Detection method, it is characterised in that：The sensitivity of high sensitivity TAL used in the method wants≤0.03
EU/mL。
6. quantified according to endotoxin content in the measure alginate material described in claim 1 or 2
Detection method, it is characterised in that：The endotoxin content of tiny electrolytic cell water used in the method is less than
0.005EU/mL。
7. quantified according to endotoxin content in the measure alginate material described in claim 1 or 2
Detection method, it is characterised in that：According to linear regression analysis, the coefficient correlation (r) of standard curve
Absolute value should be greater than or equal to 0.980, test as effective.
8. quantified according to endotoxin content in the measure alginate material described in claim 1 or 2
Detection method, it is characterised in that：The sample solution for preparing known endotoxin concns is used for interference test, surveys
Determine the rate of recovery；
When the endotoxic rate of recovery is between 50%~200%, then it is assumed that the measured value under this experimental condition
Effectively.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5551340A (en) * | 1978-10-06 | 1980-04-15 | Baxter Travenol Lab | Method of measuring concentration of endotoxin by comparison of turbidity |
| RU2325645C1 (en) * | 2006-11-13 | 2008-05-27 | Закрытое Акционерное Общество "Кдо-Тест" (Зао "Кдо-Тест") | Method of bacterial endotoxins detection with application of tal-test implying coagulogene polymer registered by structure of generated protein fractals |
| CN101387647A (en) * | 2008-09-08 | 2009-03-18 | 西王集团有限公司 | Method for detecting anhydrous dextrose bacteria endotoxin content by spectrophotometry |
| CN101718709A (en) * | 2009-11-23 | 2010-06-02 | 蚌埠丰原涂山制药有限公司 | Method for detecting bacterial endotoxin of xylitol injection |
| CN101831002A (en) * | 2009-03-11 | 2010-09-15 | 中国科学院大连化学物理研究所 | Preparation method of sodium alginate for tissue engineering |
-
2015
- 2015-12-10 CN CN201510920871.XA patent/CN106872463A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5551340A (en) * | 1978-10-06 | 1980-04-15 | Baxter Travenol Lab | Method of measuring concentration of endotoxin by comparison of turbidity |
| RU2325645C1 (en) * | 2006-11-13 | 2008-05-27 | Закрытое Акционерное Общество "Кдо-Тест" (Зао "Кдо-Тест") | Method of bacterial endotoxins detection with application of tal-test implying coagulogene polymer registered by structure of generated protein fractals |
| CN101387647A (en) * | 2008-09-08 | 2009-03-18 | 西王集团有限公司 | Method for detecting anhydrous dextrose bacteria endotoxin content by spectrophotometry |
| CN101831002A (en) * | 2009-03-11 | 2010-09-15 | 中国科学院大连化学物理研究所 | Preparation method of sodium alginate for tissue engineering |
| CN101718709A (en) * | 2009-11-23 | 2010-06-02 | 蚌埠丰原涂山制药有限公司 | Method for detecting bacterial endotoxin of xylitol injection |
Non-Patent Citations (3)
| Title |
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| 叶丹玲: "《药品生物检定技术》", 28 February 2014, 浙江大学出版社 * |
| 周国安等: "《生物制品生产规范与质量控制》", 30 April 2004, 化学工业出版社 * |
| 顾其胜等: "《海藻酸盐基生物医用材料与临床医学》", 30 April 2015, 上海科学技术出版社 * |
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Application publication date: 20170620 |