CN1217191C - Homologous system model method for quantitatively detecting bacterial endotoxin of blood - Google Patents
Homologous system model method for quantitatively detecting bacterial endotoxin of blood Download PDFInfo
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- CN1217191C CN1217191C CN 03126794 CN03126794A CN1217191C CN 1217191 C CN1217191 C CN 1217191C CN 03126794 CN03126794 CN 03126794 CN 03126794 A CN03126794 A CN 03126794A CN 1217191 C CN1217191 C CN 1217191C
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Abstract
The present invention relates to a method for the quantitative detection of endotoxins. The method for the quantitative detection of endotoxins is the same as a system model method. The technical problem to be solved is a difference, (namely interference) resulted from the differences of systems and properties. The system model method provides a new notion for solving the interference of samples of the kind to BET, which means that except the known factor such as the interference of samples of the kind to BET (G-factor bypass influence for example), other unknown factors are all taken into consideration in the overall system, and the combined influences of the factors are taken as the deviation of the overall system. As long as the deviation of the system is eliminated, the interference of samples of the kind to BET. In a certain reaction system, a digital model is established, and a sample is detected in the same system so as to eliminate difference (namely interference) resulted from the difference of system properties.
Description
Technical field
The present invention relates to the bacterial endotoxin quantitative detecting method.
Background technology
Bacterial endotoxin is one of composition of gram-negative bacterial cell wall, is a kind of main pyrogen.Detection of bacterial endotoxin has obtained more and more widely application at modern medicine and medical industry.
The employed reagent of detection of bacterial endotoxin is tachypleus amebocyte lysate (TAL or LAL).Contain factor C, factor B, proclotting enzyme and the coagulagen that can be activated by the denier bacterial endotoxin among the TAL.(temperature, pH value and noiseless material etc.) under optimum conditions, bacterial endotoxin can make TAL produce agglutinating reaction.
The methodology of detection of bacterial endotoxin is classified as follows:
I. limit detection method (gel method)
II. detection by quantitative method
1)
2)
Bacterial endotoxin detection by quantitative ratio juris is to use photoelectric detector, turbidity/colourity change transitions that endotoxin and TAL agglutinating reaction are produced is the variation of optical density (OD value), set up the mathematical model (being typical curve) of endotoxin concns and OD value and correlation parameter relation, thereby measure the endotoxin content of various samples quantitatively.In the method process, most crucial steps is correctly to set up typical curve.
Below be example with the dynamic time nephelometry, introduce the method for setting up typical curve with prior art.
Get (bottle) standard endotoxin, tire by sign and check that with bacterial endotoxin water (W) is diluted at least three geometric ratio concentration C to it
1, C
2And C
3, react on photoelectric detector with TAL respectively, obtain three corresponding dynamic response curve S
1, S
2And S
3, for the accuracy of testing, each concentration-response repeats two test tubes at least.If the numerical value of an optical density decay is set artificially, such as 92%OD, just can obtain each standard endotoxin concns (C) and TAL reaction, make optical density decay to 92%OD needed time (T).As shown in Figure 1.
Parameter T
1-C
1, T
2-C
2And T
3-C
3Retouch respectively on log-log coordinate (LogT-LogC), obtain three coordinate points.Do regretional analysis to these 3, just obtain a straight line logT=b-klogC.We claim that this straight line is the typical curve of TAL and the reaction of standard endotoxin, sees Fig. 2.Here it is the reaction time (T) and the mathematical model of endotoxin concns (C) relation, has this model just can detect unknown endotoxin concns quantitatively.The sample (S) and TAL reaction of one unknown endotoxin content are for example arranged, obtain a dynamic response curve S x, the reaction time that reaches 92%OD is Tx, as shown in Figure 3.Utilize the mathematical model of having set up (C-T typical curve) just can obtain the endotoxin content Cx of sample S quantitatively, see Fig. 4.
Existing endotoxin quantitative detecting method exists a serious defective: promptly be that water (W) is diluted to needed concentration to the standard endotoxin during preparation standard curve, then with TAL reaction (is that T/E reacts with the reaction of calling endotoxin and TAL in the following text).That is to say that T/E reaction is to be to carry out in the system of medium with water.But when the endotoxin of test sample, the T/E reaction is to carry out in the system that is medium with the sample, if exist the factor of disturbing the T/E reaction this moment in the sample system, reaction result will be variant with the reaction result in the W system.In general, T/E is reflected at and carries out in the sample system and carry out variantly in the W system, mainly shows following two aspects:
1, the difference of reaction velocity
Be example with the endotoxin that uses the dynamic time nephelometry to detect human plasma.
1.1T/E be reflected at the result who carries out in the W system
Selection (is designated as E with standard endotoxin concns 0.05,0.01 and the 0.002Eu/ml that W dilutes three good geometric ratios
0.05, E
0.01And E
0.002) react on photoelectric detector with TAL respectively, each concentration repeats two reaction tubes, does two pipe negative controls simultaneously.The corresponding dynamic response curve is seen Fig. 5 E
a, E
b, E
cAnd E
N
1.2T/E be reflected at the result who carries out in sample (S) system
Get a human normal plasma (S), be correspondingly processed after diluting by 1: 10 with W, as medium, be designated as S with this sample
10Get the standard endotoxin, be diluted to three with S and wait specific concentration 0.05,0.01 and 0.002Eu/ml (to be designated as S
10E
0.05, S
10E
0.01, S
10E
0.002), to react on photoelectric detector with above-mentioned TAL respectively with lot number, each concentration repeats two reaction tubes, does two pipe S contrasts simultaneously.The corresponding dynamic response curve is seen Fig. 5 S
a, S
b, S
cAnd S
N
Compare E
NAnd S
NCan find out that itself does not contain the endotoxin that can survey.
Compare E
a/ S
a, E
b/ S
b, E
c/ S
cCan find out that the reaction velocity of standard endotoxin in two kinds of systems of same concentration is different, endotoxin concns is low more, and the difference of reaction velocity (being reaction time T) is big more.
2, the difference of reaction tendency
Still be example with the endotoxin that uses the dynamic time nephelometry to detect human plasma.
E among Fig. 6
a, E
bAnd E
cBe the dynamic response curve of standard endotoxin and TAL, S is the dynamic response curve of certain blood samples of patients sample and TAL.Introducing us and know from the front: 1) pad value of optical density is artificial the setting, for example, can establish 92%OD, also can establish 90%OD, and key is that the OD pad value got when test sample must be consistent when setting up typical curve.2) typical curve of dynamic time nephelometry is expression reaction time (T) and the mathematical model of endotoxin concns (C) relation, and promptly the endotoxin that decides institute's test sample product according to the speed of reaction time T how much.
In Fig. 6, if we are made as 92% to the OD pad value, as can be seen, the reaction time of sample is and E
aIdentical, endotoxin content and the standard endotoxin E of this expression sample S
aShould equate.If but we are set to 85% to the OD pad value, as can be seen, the reaction time of S is and E
bIdentical, this means endotoxin content and the standard endotoxin E of S
bEquate! Why same S gets OD pad value difference, have different endotoxin contents? reason is that T/E is reflected at due to the reaction tendency difference of carrying out in two kinds of systems! Relatively their dynamic response curve can obviously be found out the trend difference of each curve.
Above-mentioned comparative analysis has shown the major defect of existing bacterial endotoxin quantitative detecting method, and promptly the mathematical model of setting up with a reaction system goes to weigh the reaction of another different systems! This is very irrational.
Summary of the invention
Bacterial endotoxin quantitative detecting method-homologous ray the modelling of blood of the present invention, the technical matters that solve are the difference (promptly disturbing) that is caused by the system different in kind.
Bacterial endotoxin quantitative detecting method-homologous ray the modelling of blood of the present invention, this method is to set up typical curve in the sample system, goes to calculate the result of tachypleus amebocyte lysate and example reaction with this typical curve, obtains the endotoxin content of sample quantitatively.
The method and the step of this modelling are as follows:
(1), use normative reference endotoxin (RSE) or international standard endotoxin (ISE) or national standard endotoxin (NSE) or working stamndard endotoxin (CSE) as standard endogenous toxic material preparation standard curve usually; Should redissolve and dilute by its request for utilization during use standard endotoxin;
(2), determine the needed standard endotoxin concns of preparation standard curve series, this series will have three dilute concentrations at least;
(3), the sample thief dilute with water is prepared into detectable concentration solution, such solution umber equates with the concentration number of the standard endotoxin series of step (2); Every part of solution adds the standard endotoxin in dilution, the concentration that makes sample solution is detectable concentration, and the endotoxic concentration of contained standard is one of standard endotoxin series concentration of step (2).Need prepare 1 part in addition and not contain the endotoxic detectable concentration sample solution of standard;
(4), the sample solution of (3) described preparation is set by step handled accordingly;
(5), getting tachypleus amebocyte lysate reacts on photoelectric detector with the sample solution of (3) set by step and (4) described preparation respectively;
(6), the response data of step (5) gained is done regretional analysis, set up typical curve;
(7), use the typical curve set up to go to detect to read blood sample of the same race to handle the data that back and tachypleus amebocyte lysate are reacted on photoelectric detector at same detectable concentration and through similarity condition, thereby obtain the endotoxin content of sample.
The term harmonization that the condition of preparation standard curve and blood sample detect.
Use all bacterial endotoxin quantitative detecting methods to detect the blood endotoxin, comprise terminal point nephelometry, dynamic turbidimetric, terminal colorimetric analysis and dynamic colourimetry.
General sample all only is the interference of one or both factors to the interference of BET, as some composition, and ion concentration, Ph values etc. can take the specific aim measure more easily to eliminate to these disturbing factors, but blood and blood product are multifactorial to the interference of BET.Complicated combined action makes the increased response except activating the G-factor bypass, and some composition has had strong inhibitory effects to BET.For example people attempt to eliminate human serum albumin (HSA) to the interference of BET but effect is undesirable always, and reason is that we always wish to take the specific aim measure to eliminate HSA how the factor of BET interference is had how many kinds of and their interference mechanism.The native system modelling proposes one and solves the new concept that this class sample disturbs BET, i.e. except the factor of known this class sample (as G-factor bypass influence) to the BET interference, other X factor is completely included in total system to be considered, the combined influence of factors is regarded as total system generation deviation, as long as eliminate system deviation, just eliminated of the interference of this class sample to BET.In certain reaction system, set up mathematical model, and in identical system test sample, make owing to difference that the system different in kind causes (promptly disturbing) is eliminated.
Description of drawings
Fig. 1 is the graph of a relation in endotoxin concns and reaction time;
Fig. 2 is the C-T canonical plotting;
Fig. 3 is a sample dynamic response curve map;
Fig. 4 is the time that must be reached default OD value by Fig. 3, reads the endotoxin concns C of testing sample by typical curve
x
Fig. 5 is that T/E is reflected at the comparison diagram that carries out in two individual system;
Fig. 6 is a comparison diagram of setting up the dynamic response curve of the dynamic response curve of typical curve and sample;
Fig. 7 is to be the T/E reaction dynamic curve diagram of medium with the sample;
Fig. 8 is the canonical plotting that is obtained by Fig. 7;
Fig. 9 is the endotoxic dynamic response curve map of TAL and standard;
Figure 10 is the canonical plotting that is obtained by Fig. 9;
Figure 11 is the reaction dynamic curve diagram of sample A;
Figure 12 is the reaction dynamic curve diagram of sample B.
Embodiment
1, it is as follows to set up the method step of homologous ray model:
A) determine to set up the needed standard endotoxin concns of typical curve.E for example
0.05, E
0.01And E
0.002.
B) getting (bottle) standard endotoxin (RSE, ISE, NSE or CSE) mixes standby by operation instruction redissolution and vortex.
C) determine the detectable concentration of sample.For example the detectable concentration of blood sample is 1: 10 dilution (S
10).
D) use the sample of detectable concentration that the standard endotoxin is diluted to required concentration.The S of dynamic time nephelometry for example
10E
0.05, S
10E
0.01And S
10E
0.002Also can release mutually and obtain containing the endotoxic detectable concentration sample of desired concn with sample (S) and standard endotoxin.Prepare portion in addition and do not contain the endotoxic detectable concentration sample of standard S
10, if necessary, S
10E
0.05, S
10E
0.01And S
10E
0.002And S
10Be correspondingly processed.
E) get TAL and react on photoelectric detector with the sample for preparing respectively, as S
10E
0.05, S
10E
0.01, S
10E
0.002And S
10, each sample repeats two reaction tubes at least.Need to establish two negative control pipes in addition, the dynamic response curve is seen Fig. 7, and the typical curve of reaction is seen Fig. 8.
F) experimental data is done regretional analysis, obtain a typical curve.LogT=KlogC for example, expression T/E are reflected at the mathematical model of reaction time (T) of carrying out in the detectable concentration sample and endotoxin concns (C) relation.
The mathematical model of setting up with said method is measured the blood sample with the unknown endotoxin content of kind, just can eliminate aforesaid reaction velocity difference, reaction tendency difference and all interference phenomenons that caused by architectural difference.
2, points for attention
It is consistent when the processing of sample must be with daily detection when a) setting up the homologous ray model.Need first heat treated as plasma sample before daily detection, the sample when then setting up model just reacts with TAL also should make heat treated after adding the standard endotoxin after.Such as other processing mode,, also require consistent as chloroform extracting, perchloric acid method etc.
The detectable concentration of sample was consistent when the sample detection concentration when b) setting up the homologous ray model must be with daily detection.As above-mentioned sample detection concentration when setting up the human plasma system model is 1: 10 dilution S
10, the sample detection concentration when daily detection also should be 1: 10 dilution S
10
C) sample that is used to set up the homologous ray model self endotoxin content under the detectable concentration state is few more good more, does not preferably contain and can survey endotoxin (as above-mentioned dynamic time method, i.e. S
10Response curve in 60 minutes, do not intersect with the OD that sets decay line).The sample that endotoxin content is high is not suitable for being used for the preparation standard curve.Detect for human blood, just use healthy human blood's sample to prepare the mark curve.
When d) typical curve of setting up with the homologous ray modelling is applied to various bacterial endotoxin quantitative detecting method, as the terminal point nephelometry, when terminal colorimetric analysis, dynamic turbidimetric, dynamic colourimetry, must follow the specific requirement of the whole bag of tricks equally, require or the like as linearity requirement, the recovery.
3, the application example of homologous ray modelling---human blood bacterial endotoxin detection by quantitative
3.1 experiment material
1) be used to set up the sample of typical curve: healthy human blood's portion is designated as S;
2) the testing sample blood samples of patients is two parts, is designated as A and B respectively;
3) standard endotoxin (CSE), tachypleus amebocyte lysate (TAL) and experimental water (W) all use Zhanjiang Andusi Biology Co., Ltd.'s product;
4) photoelectric detector;
3.2 experimental technique and step
1) gets each 1ml of blood sample, place the apyrogeneity test tube respectively, isolate blood plasma (being designated as S, A and B equally) then with the centrifugal 5-15 of 500-1500rpm minute;
2) get CSE1 and prop up, press operation instructions dissolving and vortex and mix;
3) with W S is done dilution in 1: 10 and (be designated as S
10), in dilution, add CSE, make S
10The CSE concentration that contains respectively is 0.002,0.01, and 0.05Eu/ml (is designated as E
0.002, E
0.01And E
0.05), i.e. S
10E
0.002, S
10E
0.01, S
10E
0.05, need to prepare a S simultaneously
10
4) with W A and B are done dilution in 1: 10 respectively, be designated as A
10And B
10Simultaneously, prepare A according to the method in the step 3)
10E
0.01And B
10E
10
5) being S
10E
0.002, S
10E
0.01, S
10E
0.05, S
10, A
10, A
10E
0.01, B
10, and B
10E
0.01Insert in the 65-85 ℃ of water-bath and heated 5-15 minute;
6) get TAL by the operating process of bacterial endotoxin inspection respectively with above-mentioned processing after sample on photoelectric detector, react.
3.3 experimental result
1) the endotoxic dynamic response curve of standard is seen Fig. 9
2) typical curve is seen Figure 10
3) response curve of sample A and sample B is seen Figure 11 and Figure 12
4) examining report sees Table 1
Table 1:EDS-99 bacterial endotoxin is measured system measurement report-dynamic turbidimetric
The date of inspection: 010724PM checker: FJJ minute: 15:53:00
Tachypleus amebocyte lysate lot number: 0101120 sensitivity of the limulus reagent: 0.03EU/ml temperature of reactor: 37 ℃
BET water lot number: 0106260 standard endotoxin lot number: EC-6 room temperature: 26 ℃
| The pipe number | Title | The sample lot number | Extension rate | Endotoxin (EU/ml) | The recovery | Marginal time (second) | Endotoxin measured value (EU/ml) | Standard variance SD | Coefficient of variation CV |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 | Sample-Blood sample-Blood endotoxin standard endotoxin standard endotoxin standard endotoxin standard endotoxin standard endotoxin standard negative control negative control sample-Blood sample-Blood sample-Blood sample-Blood sample-Blood sample-Blood sample-Blood sample-Blood | 7 7 7 7 7 7 7 7 ---- ---- A A A A B B B B | ?10 ?10 ?---- ?---- ?---- ?---- ?---- ?---- ?---- ?---- ?10 ?10 ?10 ?10 ?10 ?10 ?10 ?10 | 0.002 0.002 0.01 0.01 0.05 0.05 0.01 0.01 0.01 0.01 | 0.8882 0.8882 0.9082 0.9082 | ＞3600 ＞3600 2708 2626 1710 1699 1082 1052 ＞3600 ＞3600 ＞3600 ＞3600 1732 1694 ＞3600 ＞3600 1700 1706 | ?＜0.0071 ?＜0.0071 ?＜0.0007 ?＜0.0007 ?＜0.0071 ?＜0.0071 ?0.0959 ?0.0959 ?＜0.0071 ?＜0.0071 ?0.0979 ?0.0979 | ?0.00 ?57.98 ?7.78 ?21.21 ?0.00 ?0.00 ?26.87 ?0.00 ?4.24 | 0.00 2.17 0.46 1.99 0.00 0.00 1.57 0.00 0.25 |
3.4 interpretation of result
1) sizing technique BET requires: the correlation coefficient r of a. typical curve＞0.980; B. the recovery requires between 50%-200%; The endotoxin content of c.NC (negative control) is less than the minimum point concentration of typical curve.
2) related coefficient of this experimental standard curve | r|=0.99992, can be found out that by testing result table 1 recovery of sample A is 88.82%, the recovery of sample B is 90.82%, the endotoxin content of NC is 0.0007EU/ml, less than the minimum point concentration 0.002EU/ml of typical curve.So this experiment is effective.
Claims (2)
1, a kind of homologous ray modelling of bacterial endotoxin detection by quantitative of blood, this method is to set up typical curve in human normal plasma sample media system, go to calculate the result of tachypleus amebocyte lysate and plasma sample to be measured reaction with this typical curve, obtain the endotoxin content of plasma sample to be measured quantitatively;
The method and the step of this modelling are as follows:
(1), use normative reference endotoxin or international standard endotoxin or national standard endotoxin or working stamndard endotoxin as standard endogenous toxic material preparation standard curve usually; Redissolve by its request for utilization during use standard endotoxin and dilute;
(2), determine the needed standard endotoxin concns of preparation standard curve series, this series will have three dilute concentrations at least;
(3), get human normal plasma samples with water dilution and be prepared into detectable concentration solution, such solution umber equates with the concentration number of the standard endotoxin series of step (2); Every part of solution adds the standard endotoxin in dilution, making the endotoxic concentration of contained standard is one of standard endotoxin series concentration of step (2); Need prepare 1 part of human normal plasma sample solution that does not contain the endotoxic detectable concentration of standard in addition;
(4), will be set by step all samples solution of (3) described preparation place 65-85 ℃ of water-bath heating 5-15 minute;
(5), getting tachypleus amebocyte lysate reacts on photoelectric detector with all samples solution of (4) described preparation set by step;
(6), the response data of step (5) gained is done regretional analysis, set up typical curve;
(7), use the typical curve set up to remove to calculate plasma sample to be measured at same detectable concentration and through placing 65-85 ℃ of water-bath heating to handle the data that back and tachypleus amebocyte lysate are reacted in 5-15 minute on photoelectric detector, thereby obtain the endotoxin content of plasma sample to be measured.
2, according to the described homologous ray modelling of claim 1, it is characterized in that: use all bacterial endotoxin quantitative detecting methods to detect the endotoxin of plasma sample to be measured, comprise terminal point nephelometry, dynamic turbidimetric, terminal colorimetric analysis and dynamic colourimetry.
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|---|---|---|---|
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|---|---|---|---|
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1721853B (en) * | 2004-07-12 | 2011-04-20 | 湛江安度斯生物有限公司 | Preparation method of bacterial endotoxins rapid detecting reagent case |
| JP5437660B2 (en) | 2009-02-19 | 2014-03-12 | 興和株式会社 | Coagulogen raw material, method for producing the same, method and apparatus for measuring biologically active substances derived from organisms using the same |
| CN102639054B (en) * | 2009-11-18 | 2015-02-18 | 梅斯制药两合股份有限公司 | Assay for quantifying clostridial neurotoxin |
| CN102650593A (en) * | 2012-04-24 | 2012-08-29 | 湖南迪斯生物技术有限公司 | Endotoxin fungus detector |
| CN103454235B (en) * | 2013-09-13 | 2016-04-20 | 广州康盛生物科技有限公司 | A kind of ultrasonic wave added measures the method for bacteria endotoxin content in blood plasma |
| CN103592442B (en) * | 2013-10-29 | 2016-01-20 | 王明丽 | A kind of method detecting bacteria endotoxin content in Recombinant Swine Interferon α1 freeze drying powder injection |
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