CN100383528C - Method for detecting bacterial endotoxin by detecting turbidity value - Google Patents
Method for detecting bacterial endotoxin by detecting turbidity value Download PDFInfo
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- CN100383528C CN100383528C CNB2004100208862A CN200410020886A CN100383528C CN 100383528 C CN100383528 C CN 100383528C CN B2004100208862 A CNB2004100208862 A CN B2004100208862A CN 200410020886 A CN200410020886 A CN 200410020886A CN 100383528 C CN100383528 C CN 100383528C
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- turbidity value
- bacterial endotoxin
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- endotoxin
- amebocyte lysate
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Abstract
The present invention relates to a method for detecting bacterial endotoxin by measuring turbidity values, which is characterized in that an article to be detected is mixed with a limulus reagent in an equal ratio, and the article is reacted with the limulus reagent at the temperature of 37.0(+/-)1DEG C for 60(+/-)1 minutes; the turbidity value is detected and is defined as A0 =-lg (I<t>/I0'), wherein the I'0 is the initial transmitted light intensity, and I<t> is the transmitted light intensity at the time of detection; when the turbidity value is greater than or equal to 0.055, the result is positive; when the turbidity value is smaller than 0.055, the result is negative. The present invention can be realized by a special device through measurement and has the advantages that the errors of judging the glue coagulation by manual visual observation are reduced, and the result accuracy is increased; the present invention can be used for detecting bacterial endotoxin by replacing the conventional gel method and can be used for drug quality control, production process monitoring of preparations, etc.
Description
Technical field:
The present invention relates to the assay method of bacterial endotoxin, provide a kind of especially and carried out the standard that bacterial endotoxin is checked by measuring turbidity value.
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.).
The detection for bacterial endotoxin method has rabbit test method(s), limulus test.(LimulusAmebocyte Lysate LAL) is the principle of utilizing tachypleus amebocyte lysate and endotoxin generation agglutinating reaction to limulus test, a kind of external detection method of bacterial endotoxin gradient of infection in qualitative or detection by quantitative medicine or the body blood.The concrete practice is divided into gel method and sizing technique again.
Gel method is exactly a kind of qualitative, endotoxic method of determination limit.Its principle is with endotoxin sample and tachypleus amebocyte lysate mixed in equal amounts in the test tube of special use, insulation reaction after the C factor in endotoxin and the tachypleus amebocyte lysate reacts, activates proclotting enzyme, cut off the arginine peptide bond of coagulagen ad-hoc location, produce gel thereby form coagulated protein.In the Chinese Pharmacopoeia appendix in 2000 for the regulation of bacterial endotoxins test, be exactly according to the method, be used for judging in the test sample endotoxic limit the quantity of whether up to specification, need the experimenter to estimate gel whether to occur to judge the positive and negative result, the workload of experiment own is bigger, and is prone to personal error.
Sizing technique is to utilize the principle of kinetic turbidimetric assay, promptly in endotoxin and tachypleus amebocyte lysate course of reaction, adopt its turbidity of instrumentation detection record to change, default turbidity value, time required when turbidity value changes to this preset value, the logarithm of this characteristic reaction time and the logarithm of endotoxin concns were linear as the characteristic reaction time.The method requires the good reproducibility of typical curve.
Present also clear and definite without comparison corresponding relation between gel method and two kinds of methods of sizing technique, drug control institutions and manufacturer be the main gel method that adopts in real work, relies on and manually checks, and time-consuming, effort, artificial interference factor are more.
Summary of the invention:
The object of the present invention is to provide and a kind ofly carry out the standard method that bacterial endotoxin is checked by measuring turbidity value, this method is easy and simple to handle, testing result is accurate.
The invention provides and a kind ofly carry out method of checking bacterial endotoxin, it is characterized in that by measuring turbidity value:
---test sample is mixed (volume) with the tachypleus amebocyte lysate geometric ratio, and temperature of reaction is 37.0 ± 1 ℃, and the reaction time is 60 ± 1 minutes;
---detect turbidity value, turbidity value is defined as: A
0Lg (the I of '=-
t/ I
0'), I ' wherein
0Be initial transmission light intensity, I
tTransmitted intensity during for detection;
---erosion degree value is more than or equal to 0.055 o'clock, positive result; Turbidity value is less than 0.055 o'clock, negative result.
The present invention is undertaken in the method for checking bacterial endotoxin by measuring turbidity value, and used detecting instrument is required not to be strict, and being reflected in the test tube of described test sample and tachypleus amebocyte lysate carried out, and the cumulative volume of reactant liquor is 0.2 milliliter to 0.7 milliliter.The test tube that is adopted, its internal diameter can be between 5.4 millimeters to 10.2 millimeters.The detection wavelength coverage that detects turbidity value is between 400 to 700nm.Be preferably 660nm.
The present invention is undertaken in the method for checking bacterial endotoxin by measuring turbidity value in addition, neither be very high to the requirement of reagent, and the tachypleus amebocyte lysate that is adopted, its sensitivity can be 0.015-0.5EU/ml.
The key parameter turbidity value that needs to detect of the present invention is defined as: A
0Lg (the I of '=-
t/ I
0'), I ' wherein
0Be initial transmission light intensity, I
tTransmitted intensity during for detection, turbidity value and incident intensity are irrelevant, have therefore eliminated the influence of measuring pipe size, reaction system original state.In addition, this parameter can easily be measured by a lot of existing technological means and realize, can also measure realization by special instrument, has reduced the artificial visually examine and has judged gel whether error, has improved result's accuracy; Be expected to substitute conventional gel method and carry out the inspection of bacterial endotoxin, be used for the production process monitoring of drug quality control and preparation etc.
Description of drawings:
Fig. 1 changes signal for the reaction system turbidity value.
Embodiment:
The assay method that conventional bacterial endotoxin is limited the quantity of is: at first need to carry out the test sample interference test, the bacterial endotoxin working standard is diluted to 2.0 λ, 1.0 λ, 0.5 λ and 0.25 λ concentration, each dilution is done 4 pipes (10mm internal diameter test tube), vibrate above-mentioned test tube mixing content gently, the sealing mouth of pipe is put in 37 ± 1 ℃ of water-baths.Be incubated 60 ± 2 minutes and treat observations, other gets endotoxin and checks water and test sample dilution, respectively does 2 negative control pipes.4 pipes of its maximum concentration 2.0 λ should be all positive, and 4 pipes of least concentration 0.25 λ should be all negative, and the negative control pipe is then noiseless when all negative.Can limit the quantity of according to the test sample endotoxin and select the tachypleus amebocyte lysate of suitable sensitivity, do experiment according to said method and check whether the test sample endotoxin exceeds and limit the quantity of.
The present invention carries out method of checking bacterial endotoxin by the mensuration turbidity value, on endotoxin determinator, measure the turbidity value that contains endotoxin sample and tachypleus amebocyte lysate reaction system, be 60 minutes when the reaction time, turbidity value is more than or equal to 0.055 o'clock, positive result; Turbidity value is less than 0.055 o'clock, negative result.
Embodiment 1.
The operation steps of conventional gel method is: test sample and tachypleus amebocyte lysate geometric ratio are mixed in the special-purpose test tube, and volume is 0.2ml, mixing, and the sealing mouth of pipe is put in 37 ± 1 ℃ of water-baths, be incubated 60 ± 2 minutes, and the taking-up test tube is turnback slowly, observations.When in vitro gel is indeformable, not from the positive result of tube wall slippage person; Gel can not be kept perfectly, and from the negative result of tube wall slippage person.
On endotoxin determinator, test sample and tachypleus amebocyte lysate geometric ratio are mixed in 7mm internal diameter flat based tubes, temperature of reaction is 37.1 ℃, the reaction cumulative volume is 0.3ml, sensitivity of the limulus reagent is 0.06EU/ml, and test sample is the standard endotoxin solution, and concentration is 0,0.03,0.06,0.12EU/ml, when reaching 60 minutes detection time, its corresponding turbidity value is respectively 0.005,0.024,0.055 and 0.090; This test sample and tachypleus amebocyte lysate are done parallel control according to gel method simultaneously and detected, and the result shows: turbidity value is 0.005 and 0.024 o'clock, and the result of gel method is negative; Turbidity value is 0.055 and 0.090 o'clock, and the result of gel method is positive.
Embodiment 2.
On endotoxin determinator, test sample and tachypleus amebocyte lysate geometric ratio are mixed in 5.4mm internal diameter flat based tubes, temperature of reaction is 37.2 ℃, the reaction cumulative volume is 0.3ml, sensitivity of the limulus reagent is 0.5EU/ml, test sample is for containing endotoxic deionized water, reaction time, detecting its corresponding turbidity value was 0.063 when reaching 15 minutes; This test sample and tachypleus amebocyte lysate are done parallel control according to gel method simultaneously and are detected, and the result shows: turbidity value is 0.063, and the result of gel method is positive.
Embodiment 3.
On endotoxin determinator, test sample and tachypleus amebocyte lysate geometric ratio are mixed in employing 5.4mm internal diameter flat based tubes, temperature of reaction is 37.1 ℃, the reaction cumulative volume is 0.3ml, sensitivity of the limulus reagent is 0.03EU/ml, test sample is that concentration is 0.24EU/ml standard endotoxin aqueous solution, when reaching 60 minutes detection time, its corresponding turbidity value is 0.07; This test sample and tachypleus amebocyte lysate are done parallel control according to gel method simultaneously and are detected, and the result shows: turbidity value is 0.07, and the result of gel method is positive.
Embodiment 4.
On endotoxin determinator, test sample and tachypleus amebocyte lysate geometric ratio are mixed in employing 7mm internal diameter flat based tubes, temperature of reaction is 37.1 ℃, the reaction cumulative volume is 0.5ml, sensitivity of the limulus reagent is 0.03EU/ml, test sample is for containing endotoxic deionized water, and when reached 25 minutes detection time, its turbidity value was 0.063 o'clock, adopt spectrophotometer to its 400,600 and three of 700nm detect under the wavelength and measure, absorbance is respectively 0.444,0.166 and 0.151 as a result.This test sample and tachypleus amebocyte lysate are done parallel control according to gel method simultaneously and are detected, and the result shows: turbidity value is 0.063 o'clock, and the result of gel method is positive.
Embodiment 5.
Adopt spectrophotometer, under 22. ℃ room temperature, the test tube that adopts the 10.2mm internal diameter is as the reaction assay pipe, and test sample is for containing endotoxic deionized water sample, and sensitivity of the limulus reagent is 0.5EU/ml, mensuration system volume is 0.7ml, the ratio of test sample and tachypleus amebocyte lysate is 1: 1, and the detection wavelength set is 600nm, measures its absorbance, in the time of 85 minutes, absorbance is 0.124.
On endotoxin determinator, test sample and tachypleus amebocyte lysate geometric ratio are mixed in employing 7mm internal diameter flat based tubes, temperature of reaction is 37.1 ℃, and the reaction cumulative volume is 0.7ml, and sensitivity of the limulus reagent is 0.5EU/ml, when reaching 85 minutes detection time, its corresponding turbidity value is 0.10.This test sample and tachypleus amebocyte lysate are done parallel control according to gel method simultaneously and are detected, and the result shows: turbidity value is 0.10 o'clock, and the result of gel method is positive.
Embodiment 6.
On endotoxin determinator, clindamycin phosphate sodium chloride injection and the tachypleus amebocyte lysate geometric ratio of 1.5mg/ml is mixed in 7mm internal diameter flat based tubes, temperature of reaction is 37.0 ℃, sensitivity of the limulus reagent is 0.125EU/ml, reaction volume 0.3ml, in the time of 60 minutes, measuring turbidity value is 0.004.This test sample and tachypleus amebocyte lysate are done the parallel control detection according to gel method simultaneously, and the result shows: turbidity value is 0.004 o'clock, and the result of gel method is negative.
Embodiment 7.
On endotoxin determinator, test sample and tachypleus amebocyte lysate geometric ratio are mixed in 5.4mm internal diameter flat based tubes, temperature of reaction is 37.0 ℃, the reaction cumulative volume is 0.2ml, sensitivity of the limulus reagent is 0.25EU/ml, test sample is for containing endotoxic deionized water, when reaching 60 minutes detection time, its corresponding turbidity value is 0.14; This test sample and tachypleus amebocyte lysate are done parallel control according to gel method simultaneously and are detected, and the result shows: turbidity value is 0.14 o'clock, and the result of gel method is positive.
Embodiment 7
On endotoxin determinator, test sample and tachypleus amebocyte lysate geometric ratio are mixed in 7mm internal diameter flat based tubes, temperature of reaction is 37.0 ℃, the reaction cumulative volume is 0.3ml, and sensitivity of the limulus reagent is 0.06EU/ml, and test sample is a sodium-chloride water solution, concentration is 140mM, when reaching 60 minutes detection time, its corresponding turbidity value is 0.004, and this test sample and tachypleus amebocyte lysate are done parallel control according to gel method simultaneously and detected, the result shows: turbidity value is 0.004 o'clock, and the result of gel method is negative.
Embodiment 8
On endotoxin determinator test sample and tachypleus amebocyte lysate geometric ratio are mixed in 7mm internal diameter flat based tubes, temperature of reaction is 37.5 ℃, and the reaction cumulative volume is 0.3ml, sensitivity of the limulus reagent is 0.015EU/ml, test sample is no heat source water, and when reaching 60 minutes detection time, its corresponding turbidity value is 0.003; This test sample and tachypleus amebocyte lysate are done parallel control according to gel method simultaneously and are detected, and the result shows: turbidity value is 0.003 o'clock, and the result of gel method is negative.
Claims (5)
1. one kind is carried out method of checking bacterial endotoxin by measuring turbidity value, it is characterized in that:
---test sample is mixed with the tachypleus amebocyte lysate geometric ratio, and temperature of reaction is 37.0 ± 1 ℃, and the reaction time is 60 ± 1 minutes;
---detect turbidity value, the detection wavelength coverage that detects turbidity value is between 400 to 700nm, and turbidity value is defined as: A
01g (the I of '=-
t/ I
0'), I ' wherein
0Be initial transmission light intensity, I
tTransmitted intensity during for detection;
---turbidity value is more than or equal to 0.055 o'clock, positive result; Turbidity value is less than 0.055 o'clock, negative result.
2. a kind ofly carry out method of checking bacterial endotoxin by measuring turbidity value according to claim 1 is described, it is characterized in that: being reflected in the test tube of described test sample and tachypleus amebocyte lysate carried out, and the cumulative volume of reactant liquor is 0.2 milliliter to 0.7 milliliter.
3. a kind ofly carry out method of checking bacterial endotoxin by measuring turbidity value according to claim 2 is described, it is characterized in that the tachypleus amebocyte lysate that adopted, its sensitivity is 0.0150.5EU/ml.
4. a kind ofly carry out method of checking bacterial endotoxin by measuring turbidity value according to claim 2 is described, it is characterized in that the test tube that adopted, its internal diameter is 5.4 millimeters to 10.2 millimeters.
5. a kind ofly carry out method of checking bacterial endotoxin by measuring turbidity value according to claim 1 is described, the detection wavelength that it is characterized in that detecting turbidity value is 660nm.
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| CN100383528C true CN100383528C (en) | 2008-04-23 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US8394602B2 (en) * | 2007-11-12 | 2013-03-12 | Hiroshima University | Luminescent method for measuring endotoxin |
| CN101477103B (en) * | 2009-01-05 | 2012-06-27 | 范能全 | Full-pyrogen detection method for pyrogenicsubstance content in injection |
| IT1392994B1 (en) * | 2009-02-25 | 2012-04-02 | Alifax Holding S P A | PROCEDURE FOR BACTERIOLOGICAL SURVEY ON A BIOLOGICAL SAMPLE AND ITS DEVICE |
| KR20120001755A (en) * | 2009-03-13 | 2012-01-04 | 코와 가부시키가이샤 | Method for measuring biogenous biologically active substances, a program for implementing the same, and apparatus for measuring biogenous biologically active substances |
| CN109884251B (en) * | 2019-03-18 | 2021-10-22 | 天津市宇驰检测技术有限公司 | Decoration pollution detection method |
| CN109884252B (en) * | 2019-03-18 | 2021-10-22 | 天津市宇驰检测技术有限公司 | Exhaust gas detection method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4740460A (en) * | 1984-06-27 | 1988-04-26 | Wako Pure Chemical Industries, Ltd. | Process for measuring endotoxin and apparatus used therefor |
| CN1132352A (en) * | 1994-10-31 | 1996-10-02 | 和光纯药工业株式会社 | Process for measuring amount of endotoxin or (1-3)-beta-D-glucan by kinetic turbidimetric method |
| WO2004001419A1 (en) * | 2002-06-21 | 2003-12-31 | Hemofarm Koncern A.D. Pharmaceutical And Chemical Industry | Process for determining endotoxin level in hemoglobin solutions |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4740460A (en) * | 1984-06-27 | 1988-04-26 | Wako Pure Chemical Industries, Ltd. | Process for measuring endotoxin and apparatus used therefor |
| EP0347951A2 (en) * | 1984-06-27 | 1989-12-27 | Wako Pure Chemical Industries, Ltd. | Apparatus for measuring endotoxin |
| CN1132352A (en) * | 1994-10-31 | 1996-10-02 | 和光纯药工业株式会社 | Process for measuring amount of endotoxin or (1-3)-beta-D-glucan by kinetic turbidimetric method |
| WO2004001419A1 (en) * | 2002-06-21 | 2003-12-31 | Hemofarm Koncern A.D. Pharmaceutical And Chemical Industry | Process for determining endotoxin level in hemoglobin solutions |
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