CN1815235A - Judging method for quantitative determination of endotoxin concentration in sumple - Google Patents
Judging method for quantitative determination of endotoxin concentration in sumple Download PDFInfo
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- CN1815235A CN1815235A CN 200510045847 CN200510045847A CN1815235A CN 1815235 A CN1815235 A CN 1815235A CN 200510045847 CN200510045847 CN 200510045847 CN 200510045847 A CN200510045847 A CN 200510045847A CN 1815235 A CN1815235 A CN 1815235A
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Abstract
Characters of the method are as following: carrying out series of dilution for standard substance of endotoxin and sample to be measured; in reaction with reagent of horseshoe crab, measuring diagnostic reaction time when turbidity value reaches to a prearranged turbidity; making curve from logarithm of diagnostic reaction time and logarithm of diluted concentration so as to obtain slope bs of standard curve and slope b of diluted curve; magnitude of ratio of b/bs is as basis to judge whether quantitative determination can be carried out or not; b/bs is required between 133-80%. The invention discloses method for determining concentration of endotoxin in sample to be measured quantificationally, and gives out test error calculated through standard curve. Being combined with calcimeter for endotoxin and dynamic method for comparing turbidity quantificationally, the disclosed method raised accuracy of reassured result.
Description
Technical field:
The present invention relates to endotoxic detection, the determination methods of endotoxin concns in a kind of quantitative measurement sample is provided especially.
Background technology:
Bacterial endotoxin (Bacterial Endotoxin) be gramnegative bacterium produce have different 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.(Limulus Amebocyte 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 body fluid.The concrete practice is divided into gel method again and dynamically than turbid sizing technique.Than for the turbid sizing technique, whether can come endotoxin concns in the calculation sample for dynamically,, provide a rational criterion and be very important to improve the accuracy of measurement result by typical curve.
Summary of the invention;
The object of the present invention is to provide the determination methods of endotoxin concns in a kind of quantitative measurement sample, whether can undertaken quantitatively providing error range, and then provide foundation for quantitative measurement endotoxin more accurately by typical curve with endotoxin in the judgement sample.
Particularly, the invention provides the determination methods of endotoxin concns in a kind of quantitative measurement sample, it is characterized in that:
---endotoxin standard items and testing sample are done serial dilution;
---with above-mentioned through serial dilution the endotoxin standard items and testing sample respectively with the tachypleus amebocyte lysate reaction, the characteristic reaction time when recording turbidity value reaching a default turbidity on endotoxin determinator, wherein the sensitivity of the limulus reagent scope is 0.03-0.5EU/ml;
---the logarithm of characteristic reaction time of the serial dilution endotoxin standard items that obtained and testing sample and the logarithm of dilute concentration are made curve, obtain the typical curve and the diluted sample curve of double-log form; And and then obtain the slope bs and the diluted sample slope of a curve b of typical curve;
---as the basis for estimation that can carry out quantitative measurement, require b/bs between 133-80% the size of the slope bs of typical curve and the two ratio b/bs of diluted sample rate of curve b.
In the quantitative measurement sample of the present invention in the determination methods of endotoxin concns, when b/bs is between the 133-80% time, endotoxin concns difference is in 16 times of scopes, and error at measurment is between 50-200%.
In the quantitative measurement sample of the present invention in the determination methods of endotoxin concns, when b/bs is between the 111-91% time, endotoxin concns difference is in 1024 times of scopes, and error at measurment is between 50-200%.
In the quantitative measurement sample of the present invention in the determination methods of endotoxin concns, when b/bs is between the 104-97% time, endotoxin concns difference is in 256 times of scopes, and error at measurment is between 80-120%.
In the determination methods of endotoxin concns, described default turbidity value is 0.02 in the quantitative measurement sample of the present invention.
The principle of kinetic turbidimetric assay is, in endotoxin and tachypleus amebocyte lysate course of reaction, adopt its turbidity of instrumentation detection record to change, default turbidity value 0.02, time required when turbidity value changes to this preset value 0.02 is as characteristic reaction time t0 .02, the logarithm of the logarithm of this characteristic reaction time and endotoxin concns C is linear, i.e. 1g (t0.02)=a+b 1g (C).When the endotoxin maximum concentration is C
0, when extension rate is D, C=C then
0/ D, 1g (t0.02)=a+1g (C
0)+b 1g (1/D), description standard slope of a curve should be identical with slope of standard curve just can be quantitative, variant certainly during practical measurement.
For endotoxin standard items working fluid, under different extension rate Ds1, Ds2, measure, obtain the corresponding characteristic reaction time (t0.02) respectively
1(t0.02)
21g (t0.02) then
1/ (t0.02)
2=bs 1g (Ds2/Ds1); At identical (t0.02)
1With (t0.02)
2In the scope, for sample, extension rate is D1, D2, then 1g (t0.02)
1/ (t0.02)
2=b 1g (D2/D1); Obviously, b/bs=1g (Ds1/Ds2)/1g (D1/D2), if D1/D2=α * (Ds1/Ds2), or α=(D1/D2)/(Ds1/Ds2), α is the ratio of diluted sample multiple and the ratio of the ratio of work product extension rate, or be the ratio of sample concentration and the ratio of the ratio of endotoxin standard items working fluid concentration, promptly error at measurment should be 100% under the perfect condition.
B/bs=(1g (Ds1/Ds2))/(1g (α)+1g (Ds1/Ds2)) then
Or 1g (α)=(1g (Ds1/Ds2)) ((1/ (b/bs))-1)
B/bs is the ratio of dilution of sample rate of curve and slope of standard curve, and it is relevant with Ds1/Ds2 ratio, error at measurment α.In certain measurement range, the b/bs value is more near 100%, and then error at measurment α is more near 100%; Control the b/bs value, just can control error at measurment.
The present inventor is just according to above-mentioned principle, the determination methods of endotoxin concns in the quantitative measurement sample has been proposed, quantitatively provided the test error of coming endotoxin concns in the calculation sample by typical curve, also can be with endotoxin determinator, dynamically be used than turbid sizing technique, judge the test error of quantitative measurement sample endotoxin concns, thereby improved the accuracy of measurement result.
Embodiment:
Embodiment 1
Getting sensitivity is that 0.125EU/ml, sign volume of dissolution are the tachypleus amebocyte lysate dry powder of 0.5ml, and adding 0.5ml does not have the heat source water dissolving, gets tachypleus amebocyte lysate solution; Get 160EU endotoxin standard items working fluid, press dilutability 4 proportional diluted to 1024 times, obtain the endotoxin of variable concentrations, respectively get 0.2ml in 7mm internal diameter flat based tubes, add 0.1ml tachypleus amebocyte lysate solution respectively, mix the back and insert in the endotoxin determinator detection hole, temperature of reaction is 37.1 ℃, and be 60 minutes detection time.Reaction obtains characteristic reaction time t0 .02 separately after finishing, and the logarithm of t0.02 and the logarithm of endotoxin concns are made typical curve, and slope bs is-0.200; The logarithm of t0.02 and the logarithm of extension rate inverse are done the standard dilution curve, and slope is-0.200, and bs is identical with slope of standard curve.
Get endotoxin standard items working fluid sample 1, press dilutability 4 proportional diluted to 1024 times, obtain the sample of variable concentrations, respectively get 0.2ml in 7mm internal diameter flat based tubes, add 0.1ml respectively.Tachypleus amebocyte lysate solution, reactions steps is the same, obtains characteristic reaction time t0 .02 separately.The logarithm of t0.02 and the logarithm of corresponding extension rate inverse are made the dilution curve of sample 1, slope b is-0.222, b/bs=111%, and sample 1 ratio the highest, least concentration that the establishing criteria curve calculation obtains is 512 times, actual extension rate is 1024 times, and error at measurment is 50%.
Get and contain endotoxic water sample 2, press dilutability 4 proportional diluted to 1024 times, obtain the sample of variable concentrations, respectively get 0.2ml in 7mm internal diameter flat based tubes, add 0.1ml tachypleus amebocyte lysate solution respectively, reactions steps is the same, obtain characteristic reaction time t0 .02 separately, the logarithm of t0.02 and the logarithm of corresponding extension rate inverse are made the dilution curve of sample 2, slope b is-0.182, b/bs=91%, and sample 2 ratios the highest, least concentration that the establishing criteria curve calculation obtains are 2048 times, actual extension rate is 1024 times, and error at measurment is 200%.
Embodiment 2
Getting sensitivity is that 0.125EU/ml, sign volume of dissolution are the tachypleus amebocyte lysate dry powder of 0.5ml, and adding 0.5ml does not have the heat source water dissolving, gets tachypleus amebocyte lysate solution; Get 160EU endotoxin standard items working fluid, press dilutability 2 proportional diluted to 16 times, obtain the endotoxin of variable concentrations, respectively get 0.2ml in 7mm internal diameter flat based tubes, add 0.1ml tachypleus amebocyte lysate solution respectively, mix the back and insert in the endotoxin determinator detection hole, temperature of reaction is 36.7 ℃, and be 60 minutes detection time.Reaction obtains characteristic reaction time t0 .02 separately after finishing, and the logarithm with 0.02 and the logarithm of endotoxin concns are made typical curve, slope bs is-0.200, the logarithm of t0.02 and the logarithm of extension rate inverse are done the standard dilution curve, and slope is-0.200, and bs is identical with slope of standard curve.
Get and contain endotoxic water sample 3, press dilutability 2 proportional diluted to 16 times, obtain the sample of variable concentrations, respectively get 0.2ml in 7mm internal diameter flat based tubes, add 0.1ml tachypleus amebocyte lysate solution respectively, reactions steps is the same, obtain characteristic reaction time t0 .02 separately, the logarithm of t0.02 and the logarithm of corresponding extension rate inverse are made the dilution curve of sample 3, slope b is-0.266, sample 3 ratios the highest, least concentration that b/bs=133% establishing criteria curve calculation obtains are 8 times, and actual extension rate is 16 times, and error at measurment is 50%.
Get endotoxin standard items working fluid sample 4 by dilutability 2 proportional diluted to 16 times, obtain the sample of variable concentrations, respectively get 0.2ml in 7mm internal diameter flat based tubes, add 0.1ml tachypleus amebocyte lysate solution respectively, reactions steps is the same.Obtain characteristic reaction time t0 .02 separately, the logarithm of t0.02 and the logarithm of corresponding extension rate inverse are made the dilution curve of sample 4, slope b is-0.160, b/bs=80%, sample 4 ratios the highest, least concentration that the establishing criteria curve calculation obtains are 32 times, actual extension rate is 16 times, and error at measurment is 200%.
Embodiment 3
Getting sensitivity is that 0.125EU/ml, sign volume of dissolution are the tachypleus amebocyte lysate dry powder of 0.5ml, and adding 0.5ml does not have the heat source water dissolving, gets tachypleus amebocyte lysate solution; Get 20EU endotoxin standard items working fluid, press dilutability 4 proportional diluted to 256 times, obtain the endotoxin of variable concentrations, respectively get 0.2ml in 7mm internal diameter flat based tubes, add 0.1ml tachypleus amebocyte lysate solution respectively, mix the back and insert in the endotoxin determinator detection hole, temperature of reaction is 37.1 ℃, and be 60 minutes detection time.Reaction obtains characteristic reaction time t0 .02 separately after finishing, and the logarithm of t0.02 and the logarithm of endotoxin concns are made typical curve, and slope bs is-0.170; The logarithm of t0.02 and the logarithm of extension rate inverse are done the standard dilution curve, and slope is 0.170, and bs is identical with slope of standard curve.
Get endotoxin standard items working fluid sample 5 by dilutability 4 proportional diluted to 256 times, obtain the sample of variable concentrations, respectively get 0.2ml in 7mm internal diameter flat based tubes, add 0.1ml tachypleus amebocyte lysate solution respectively, reactions steps is the same.Obtain characteristic reaction time t0 .02 separately, the logarithm of t0.02 and the logarithm of corresponding extension rate inverse are made the dilution curve of sample 5, slope b is-0.176, sample 5 ratios the highest, least concentration that b/bs=104% establishing criteria curve calculation obtains are 204.8 times, actual extension rate is 256 times, and error at measurment is 80%.
Get endotoxin standard items working fluid sample 6, press dilutability 4 proportional diluted to 256 times, obtain the sample of variable concentrations, respectively get 0.2ml in 7mm internal diameter flat based tubes, add 0.1ml tachypleus amebocyte lysate solution respectively, reactions steps is the same.Obtain characteristic reaction time t0 .02 separately, the logarithm of t0.02 and the logarithm of corresponding extension rate inverse are made the dilution curve of sample 6, slope b is-0.165, sample 6 ratios the highest, least concentration that b/bs=97% establishing criteria curve calculation obtains are 307.2 times, actual extension rate is 256 times, and error at measurment is 120%.
Embodiment 4
Getting sensitivity is that 0.5EU/ml and 0.03EU/ml sign volume of dissolution are the tachypleus amebocyte lysate dry powder of 0.5ml, and adding 0.5ml does not have the heat source water dissolving, gets the tachypleus amebocyte lysate of different sensitivity; Get 200EU endotoxin standard items working fluid, press dilutability 4 proportional diluted to 1024 times, obtain the endotoxin of variable concentrations, respectively get 0.2ml in 7mm internal diameter flat based tubes, the tachypleus amebocyte lysate that adds the 0.1ml different sensitivity respectively, mix the back and insert in the endotoxin determinator detection hole, temperature of reaction is 37.3 ℃, and be 60 minutes detection time.Reaction obtains characteristic reaction time t0 .02 separately after finishing, and the logarithm of t0.02 and the logarithm of endotoxin concns are made typical curve, and the slope bs of 0.5EU/ml sensitivity tachypleus amebocyte lysate is-0.2828; 0.03EU/ml the slope bs of sensitivity tachypleus amebocyte lysate is-0.2898.
Comparative example:
Use conventional endotoxin inspection technique to carry out the endotoxin limit examine, need the manual record reaction time, artificial visually examine's judged result, single people can not finish a large amount of sample detection.
The present invention utilizes the characteristics of instrument itself, writes down the characteristic reaction time of a plurality of laboratory samples automatically, the ratio of the slope bs of calculation sample dilution curve slope b and typical curve again, and experimental result is objective, error range may command, high efficiency.
Claims (6)
1, the determination methods of endotoxin concns in a kind of quantitative measurement sample is characterized in that:
---endotoxin standard items and testing sample are done serial dilution;
---with above-mentioned through serial dilution the endotoxin standard items and testing sample respectively with the tachypleus amebocyte lysate reaction, the characteristic reaction time when recording turbidity value reaching a default turbidity on endotoxin determinator, wherein the sensitivity of the limulus reagent scope is 0.03-0.5EU/ml;
---the logarithm of characteristic reaction time of the serial dilution endotoxin standard items that obtained and testing sample and the logarithm of dilute concentration are made curve, obtain the typical curve and the diluted sample curve of double-log form; And and then obtain the slope bs and the diluted sample slope of a curve b of typical curve;
---as the basis for estimation that can carry out quantitative measurement, require b/bs between 133-80% the size of the slope bs of typical curve and the two ratio b/bs of diluted sample rate of curve b.
2, according to the determination methods of endotoxin concns in the described quantitative measurement sample of claim 1, it is characterized in that: when b/bs is between the 133-80% time, endotoxin concns difference is in 16 times of scopes, and error at measurment is between 50-200%.
3, according to the determination methods of endotoxin concns in the described quantitative measurement sample of claim 1, it is characterized in that: when b/bs is between the 111-91% time, endotoxin concns difference is in 1024 times of scopes, and error at measurment is between 50-200%.
4, according to the determination methods of endotoxin concns in the described quantitative measurement sample of claim 1, it is characterized in that: when b/bs is between the 104-97% time, endotoxin concns difference is in 256 times of scopes, and error at measurment is between 80-120%.
5, according to the determination methods of endotoxin concns in the described quantitative measurement sample of claim 1, it is characterized in that: described default turbidity value is 0.02.
6, according to the determination methods of endotoxin concns in the described quantitative measurement sample of claim 1, it is characterized in that: during detection, temperature of reaction is 37.0 ± 0.3 ℃, and be 60 minutes detection time.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2407788A1 (en) * | 2009-03-13 | 2012-01-18 | Kowa Company, Ltd. | Method for measuring biogenous biologically active substances, a program for implementing the same, and apparatus for measuring biogenous biologically active substances |
| CN101477103B (en) * | 2009-01-05 | 2012-06-27 | 范能全 | Full-pyrogen detection method for pyrogenicsubstance content in injection |
| CN104155442A (en) * | 2008-08-18 | 2014-11-19 | 拜奥泰克公司 | Enhancing Endotoxin Detection |
-
2005
- 2005-02-05 CN CN 200510045847 patent/CN1815235A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104155442A (en) * | 2008-08-18 | 2014-11-19 | 拜奥泰克公司 | Enhancing Endotoxin Detection |
| CN101477103B (en) * | 2009-01-05 | 2012-06-27 | 范能全 | Full-pyrogen detection method for pyrogenicsubstance content in injection |
| EP2407788A1 (en) * | 2009-03-13 | 2012-01-18 | Kowa Company, Ltd. | Method for measuring biogenous biologically active substances, a program for implementing the same, and apparatus for measuring biogenous biologically active substances |
| CN102348984A (en) * | 2009-03-13 | 2012-02-08 | 兴和株式会社 | Method for measuring biogenous biologically active substances, a program for implementing the same, and apparatus for measuring biogenous biologically active substances |
| EP2407788A4 (en) * | 2009-03-13 | 2013-04-17 | Kowa Co | Method for measuring biogenous biologically active substances, a program for implementing the same, and apparatus for measuring biogenous biologically active substances |
| US8507282B2 (en) | 2009-03-13 | 2013-08-13 | Kowa Company, Ltd. | Method for measuring physiologically active substance of biological origin, program for implementing the same, and apparatus for measuring physiologically active substance of biological origin |
| CN102348984B (en) * | 2009-03-13 | 2014-04-02 | 兴和株式会社 | Method for measuring biogenous biologically active substances, a program for implementing the same, and apparatus for measuring biogenous biologically active substances |
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