CN1318848C - Buffer liquid for bacteria endotoxin horseshoe crab test detection method and process for preparing same - Google Patents
Buffer liquid for bacteria endotoxin horseshoe crab test detection method and process for preparing same Download PDFInfo
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- CN1318848C CN1318848C CNB2003101192674A CN200310119267A CN1318848C CN 1318848 C CN1318848 C CN 1318848C CN B2003101192674 A CNB2003101192674 A CN B2003101192674A CN 200310119267 A CN200310119267 A CN 200310119267A CN 1318848 C CN1318848 C CN 1318848C
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- endotoxin
- damping fluid
- phenol red
- tris
- detection method
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- 239000002158 endotoxin Substances 0.000 title claims abstract description 53
- 238000001514 detection method Methods 0.000 title claims abstract description 16
- 239000000872 buffer Substances 0.000 title abstract description 4
- 239000007788 liquid Substances 0.000 title description 3
- 241001529572 Chaceon affinis Species 0.000 title 1
- 238000004519 manufacturing process Methods 0.000 title 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229960003531 phenolsulfonphthalein Drugs 0.000 claims abstract description 24
- 241000239218 Limulus Species 0.000 claims abstract description 20
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical class OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000013016 damping Methods 0.000 claims description 33
- 239000012530 fluid Substances 0.000 claims description 33
- 231100000284 endotoxic Toxicity 0.000 claims description 20
- 230000002346 endotoxic effect Effects 0.000 claims description 20
- 238000004321 preservation Methods 0.000 claims description 8
- 239000007983 Tris buffer Substances 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 abstract description 7
- 239000007853 buffer solution Substances 0.000 abstract 5
- 230000000694 effects Effects 0.000 abstract 1
- 238000011084 recovery Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 6
- 239000013642 negative control Substances 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 239000003513 alkali Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000012895 dilution Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 241000239222 Tachypleus Species 0.000 description 2
- 230000004308 accommodation Effects 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 230000004523 agglutinating effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 238000007905 drug manufacturing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
一种用于细菌内毒素鲎试验检测法的缓冲液，其特征在于所述缓冲液为含有酚红的Tris-HCl缓冲液，酚红的重量浓度为0.005-0.1%。其制备方法是将酚红和Tris盐称重后，在150-200度干烤1-5小时；再加入无内毒素的水，用浓盐酸调节缓冲液的pH至所需要的值；高压消毒无菌保存或冷冻保存。本发明的缓冲液可以用来稀释被测样品，同时可以调节并指示样品pH。本发明的用于细菌内毒素鲎试验检测法的缓冲液，在两种内毒素测定法中对内毒素的测定无影响，均符合鲎试验法测定的规定。A buffer solution for bacterial endotoxin limulus test detection method, characterized in that the buffer solution is Tris-HCl buffer solution containing phenol red, and the weight concentration of phenol red is 0.005-0.1%. Its preparation method is to weigh the phenol red and Tris salt, then dry bake at 150-200 degrees for 1-5 hours; then add endotoxin-free water, adjust the pH of the buffer to the required value with concentrated hydrochloric acid; autoclave Store aseptically or freeze. The buffer solution of the invention can be used to dilute the sample to be tested, and at the same time can adjust and indicate the pH of the sample. The buffer solution used in the limulus test for detection of bacterial endotoxin of the present invention has no effect on the determination of endotoxin in the two endotoxin determination methods, and all conform to the determination requirements of the limulus test.
Description
Technical field:
The present invention relates to the limulus test detection method of bacterial endotoxin, a kind of adjusting of limulus test and damping fluid of indication sample pH value of being used for is provided especially.
Background technology:
Bacterial endotoxin (Bacterial Endotoxin) be gramnegative bacterium produce have various bioactive macromolecular substances, its main chemical compositions is lipopolysaccharides (LPS).Endotoxin is the major pollutants matter in the injection medicine (raw material etc.).
At present, the detection for bacterial endotoxin method mainly contains rabbit test method(s), limulus test.Limulus test is a mechanism of utilizing tachypleus amebocyte lysate and endotoxin generation agglutinating reaction, detects a kind of external detection method of the bacterial endotoxin that infects in medicine and the body blood in the mode of qualitative (gel method) or quantitative (dynamic turbidimetric).In a single day generally, in certain pH range (6.5-8.5), limulus test can normally be measured, but exceeds this scope, false positive or false negative can appear in measurement result.For the intermediate product in the sample that contains sodium bicarbonate, the particularly drug production process, often contain different types of buffer salt, pH often is lower than 6.5 (as acetate) or is higher than 8.5 (carbonate), this just needs constantly dilution and regulates pH with no endotoxic acid, alkali, and monitors with pH test paper or pH meter.This process is very complicated, and is difficult to accurate control, particularly introduces endotoxic pollution easily, therefore is badly in need of a kind of dilute sample that is used for, and can regulate and indicate simultaneously the damping fluid of sample pH value.
Summary of the invention:
The object of the present invention is to provide a kind of bacterial endotoxin limulus test detection method that is used for, can be used for dilute sample, can regulate and indicate simultaneously the damping fluid of sample pH value.
The invention provides a kind of damping fluid that is used for bacterial endotoxin limulus test detection method, it is characterized in that described damping fluid is to contain phenol red Tris-HCl damping fluid, phenol red weight concentration is 0.005-0.1%.
The present invention is used for the damping fluid of bacterial endotoxin limulus test detection method, described Tris-HCl damping fluid, and Tris concentration is preferably 30-500mM, and pH is at 7-8.5.
The present invention also provides the above-mentioned preparation method who is used for the damping fluid of bacterial endotoxin limulus test detection method, after it is characterized in that phenol red and Tris salt weighed, does roasting 1-5 hour at the 150-200 degree; Add no endotoxic water again, the pH that regulates damping fluid with concentrated hydrochloric acid is to needed value (being generally 7-8); Aseptic preservation of autoclave sterilization or freezing preservation.
The present invention is used for the damping fluid of bacterial endotoxin limulus test detection method, is the scope pinkiness of 6.8-8.2 at pH, can dilute sample, and the pH value of regulating sample simultaneously is in suitable scope, so that measuring endotoxin carries out in stable p H.The present invention is used for the damping fluid of bacterial endotoxin limulus test detection method, measure in the endotoxic experiment at limulus test, be used for qualitative method---during gel method, the mensuration of not having endotoxic negative control is not had influence, show positive distinctive gel reaction adding endotoxic positive control; Be used for sizing technique---during dynamic turbidimetric, the mensuration of not having endotoxic negative control there is not influence, to adding the endotoxic recovery of endotoxic positive control between 50-200%.Be the damping fluid that the present invention is used for bacterial endotoxin limulus test detection method, in two kinds of endotoxin measurement methods, measuring endotoxin do not had influence, all meet the regulation that limulus test is measured.
Embodiment:
Embodiment 1. is used for the typical curve of endotoxin measurement
On endotoxin determinator, based on typical curve LogT
(OD0.02)=a+b Log (c) adopts standard endotoxin concns 0.22-100EU/ml, tests with the tachypleus amebocyte lysate of 0.06EU/ml sensitivity, and obtaining corresponding standard curve is LogT
(oD0.02)=-0.4129Log (c)+3.2038, wherein T
(OD0.02)Be the characteristic reaction time, c is an endotoxin concns.
Embodiment 2. contains the preparation 1 of phenol red Tris-HCl damping fluid
0.005 gram is phenol red, 0.363 gram Tris salt is weighed and is placed in the clean vial, does roasting 200 degree 1 hour, to eliminate endotoxin wherein; Add 100 milliliters of no endotoxic water, regulate pH to 7.0 with concentrated hydrochloric acid, aseptic preservation of autoclave sterilization or freezing preservation are standby.
Embodiment 3. contains the preparation 2 of phenol red Tris-HCl damping fluid
0.1 gram is phenol red, 6.05 gram Tris salt are weighed and are placed in the vial, do roasting 150 degree 5 hours, to eliminate endotoxin wherein; Add 100 milliliters of no endotoxic water, regulate pH to 8.5 with concentrated hydrochloric acid, aseptic preservation of autoclave sterilization or freezing preservation are standby.
Embodiment 4. phenol red concentration are tested
The Tris-HCl damping fluid of preparation 250mM pH7.6, containing phenol red concentration respectively is 0,0.005,0.01,0.05,0.1%, carries out the measuring endotoxin experiment.Do not add endotoxic negative control result and be all feminine gender.The endotoxin that adds same concentrations, the endotoxin concns that records on endotoxin determinator is respectively 73.0,95.6,89.2,110.2,95.6EU/ml, the endotoxic recovery is respectively 100,131,122,151,131%, meets the regulation of the recovery between 50-200%.Contain phenol red damping fluid, with acid-alkali accommodation its pH value, when pH less than 6.8 the time, be yellow; When pH greater than 8.2 the time, take on a red color; When pH is between 6.8-8.2, be orange red.
The experiment of embodiment 5.Tris salinity
To contain phenol red concentration be 0.01% in preparation, the Tris-HCl damping fluid of pH7.5, and the Tris salinity is respectively 30,63,125,250,500mM, carries out the measuring endotoxin experiment.Do not add endotoxic negative control result and be all feminine gender.The endotoxin 3.8EU/ml that adds same concentrations respectively, the endotoxin concns that records on endotoxin determinator is respectively 3.8,6.45,4.98,5.35,6.4EU/ml, the endotoxic recovery is respectively 100,170,131,141,168%, meets the regulation of the recovery between 50-200%.Contain phenol red damping fluid, with acid-alkali accommodation its pH value, when pH less than 6.8 the time, be yellow; When pH greater than 8.2 the time, take on a red color; When pH is between 6.8-8.2, be orange red.
The pH experiment of embodiment 6.Tris-HCl damping fluid
It is 0.01% that preparation contains phenol red concentration, the Tris-HCl damping fluid of 250mM, and pH is respectively 7.0,7.5,8.0,8.5, carries out the measuring endotoxin experiment.Do not add endotoxic negative control result and be all feminine gender.The endotoxin that adds same concentrations 4.2EU/ml respectively is with reference to product, the endotoxin concns that records on endotoxin determinator is respectively 3.85,5.35,5.76,4.81EU/ml, the endotoxic recovery is respectively 92,139,150,125%, meets the regulation of the recovery between 50-200%.Contain phenol red damping fluid, regulate its pH value with concentrated acid alkali, when pH less than 6.8 the time, be yellow; When pH greater than 8.2 the time, take on a red color; When pH is between 6.8-8.2, be orange red.
Measuring endotoxin in embodiment 7. acetate buffer solutions
To the sample that contains 0.5% human serum albumins (being dissolved in the acetate buffer solution of 20mM pH3.8), the endotoxin that adds 9.81EU/ml, it is 0.01% that employing contains phenol red concentration, the Tris-HCl damping fluid of 150mM pH7.8 dilutes, solution is orange red after diluting 4 times, recording endotoxin content is 9.38EU/ml, and the endotoxic recovery is 96%.
Measuring endotoxin in the embodiment 8. sodium bicarbonate buffer liquid
The sodium bicarbonate buffer liquid of preparation 20mM pH10, the endotoxin that adds 10.7EU/ml, it is 0.01% that employing contains phenol red concentration, the Tris-HCl damping fluid of 250mM pH7.7 dilutes, after diluting 2 times, it is orange red that solution is, and recording endotoxin content is 12.45EU/ml, and the endotoxic recovery is 116%.
The application of embodiment 9. phenol red Tris-HCl damping fluids in gel method mensuration endotoxin
It is 0.01% that employing contains phenol red concentration, the Tris-HCl damping fluid of 250mM pH7.7, and as the dilution of sample,
With damping fluid: detected water sample=ratio of 1: 1 is carried out the dilution of detected water sample product, adopts gel method to measure endotoxin content in the detected water sample product, does feminine gender simultaneously, positive control is tested.The negative mensuration of result do not have influence, and positive mensuration shows positive distinctive gel reaction.
Claims (2)
1, a kind of damping fluid that is used for bacterial endotoxin limulus test detection method is characterized in that described damping fluid is to contain phenol red Tris-HCl damping fluid, and phenol red weight concentration is 0.005-0.1%, and Tris concentration is 30-500mM, and pH is 7-8.5.
2, the described preparation method who is used for the damping fluid of bacterial endotoxin limulus test detection method of a kind of claim 1 after it is characterized in that phenol red and Tris salt weighed, did roasting 1-5 hour at the 150-200 degree; Add no endotoxic water again, the pH that regulates damping fluid with concentrated hydrochloric acid is to needed value; Aseptic preservation of autoclave sterilization or freezing preservation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101192674A CN1318848C (en) | 2003-11-26 | 2003-11-26 | Buffer liquid for bacteria endotoxin horseshoe crab test detection method and process for preparing same |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2003101192674A CN1318848C (en) | 2003-11-26 | 2003-11-26 | Buffer liquid for bacteria endotoxin horseshoe crab test detection method and process for preparing same |
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| Publication Number | Publication Date |
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| CN1621842A CN1621842A (en) | 2005-06-01 |
| CN1318848C true CN1318848C (en) | 2007-05-30 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP5403516B2 (en) * | 2007-11-12 | 2014-01-29 | 国立大学法人広島大学 | Endotoxin concentration measurement method and concentration measurement kit |
| CN105136544A (en) * | 2015-10-10 | 2015-12-09 | 青岛华仁医药包装材料科技有限公司 | Bacterial endotoxin detection method for multi-layer co-extrusion infusion film |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0370700A1 (en) * | 1988-11-25 | 1990-05-30 | Sensititre Limited | Process for handling horseshoe crab amoebocyte lysate |
| CN1105757A (en) * | 1993-02-26 | 1995-07-26 | 生化学工业株式会社 | Reagent for endotoxin assay and method for endotoxin assay using the same |
| US5702882A (en) * | 1993-09-30 | 1997-12-30 | Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation) | Reagent for endotoxin-specific assay |
| WO2003002976A2 (en) * | 2001-06-28 | 2003-01-09 | Biowhittaker, Inc. | Methods and reagents for detecting endotoxin |
-
2003
- 2003-11-26 CN CNB2003101192674A patent/CN1318848C/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0370700A1 (en) * | 1988-11-25 | 1990-05-30 | Sensititre Limited | Process for handling horseshoe crab amoebocyte lysate |
| CN1105757A (en) * | 1993-02-26 | 1995-07-26 | 生化学工业株式会社 | Reagent for endotoxin assay and method for endotoxin assay using the same |
| US5702882A (en) * | 1993-09-30 | 1997-12-30 | Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation) | Reagent for endotoxin-specific assay |
| WO2003002976A2 (en) * | 2001-06-28 | 2003-01-09 | Biowhittaker, Inc. | Methods and reagents for detecting endotoxin |
| US20030054432A1 (en) * | 2001-06-28 | 2003-03-20 | Biowhittaker, Inc. | Methods and reagents for detecting endotoxin |
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| CN1621842A (en) | 2005-06-01 |
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