CN1721853B - Preparation method of bacterial endotoxins rapid detecting reagent case - Google Patents
Preparation method of bacterial endotoxins rapid detecting reagent case Download PDFInfo
- Publication number
- CN1721853B CN1721853B CN 200410028071 CN200410028071A CN1721853B CN 1721853 B CN1721853 B CN 1721853B CN 200410028071 CN200410028071 CN 200410028071 CN 200410028071 A CN200410028071 A CN 200410028071A CN 1721853 B CN1721853 B CN 1721853B
- Authority
- CN
- China
- Prior art keywords
- amebocyte lysate
- endotoxin
- reaction
- tachypleus amebocyte
- sensitivity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 239000002158 endotoxin Substances 0.000 title claims abstract description 79
- 238000002360 preparation method Methods 0.000 title claims description 14
- 239000003153 chemical reaction reagent Substances 0.000 title description 3
- 238000000034 method Methods 0.000 claims abstract description 33
- 230000035484 reaction time Effects 0.000 claims abstract description 27
- 241000239222 Tachypleus Species 0.000 claims description 43
- 239000006166 lysate Substances 0.000 claims description 43
- 238000001514 detection method Methods 0.000 claims description 25
- 230000035945 sensitivity Effects 0.000 claims description 24
- 238000012360 testing method Methods 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 14
- 238000002474 experimental method Methods 0.000 claims description 8
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- 231100000284 endotoxic Toxicity 0.000 claims description 4
- 230000002346 endotoxic effect Effects 0.000 claims description 4
- 238000012856 packing Methods 0.000 claims description 4
- 238000002834 transmittance Methods 0.000 claims description 4
- 239000003643 water by type Substances 0.000 claims description 3
- 230000001143 conditioned effect Effects 0.000 claims description 2
- 238000003825 pressing Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 10
- 238000005516 engineering process Methods 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 abstract 3
- 239000003053 toxin Substances 0.000 abstract 1
- 231100000765 toxin Toxicity 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 239000002510 pyrogen Substances 0.000 description 3
- 108090000056 Complement factor B Proteins 0.000 description 1
- 102000003712 Complement factor B Human genes 0.000 description 1
- 230000004523 agglutinating effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 108010048121 pro-clotting enzyme Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011046 pyrogen test Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a method for preparing bacterial endotoxin fast measuring agent box in the field of bacterial endotoxin measuring technology. It can cut down bacterial endotoxin measuring time according to the inverse relation of agent activity and reaction time, and the inverse relation of toxin density and reaction time. The bacterial endotoxin fast measuring agent box can cut off the bacterial endotoxin measuring reaction time of gelling process form 60 minutes to 20 minutes or shorter.
Description
Technical field
The present invention relates to the detection of bacterial endotoxin technical field, the preparation method of the quick detection kit of particularly a kind of bacterial endotoxin.
Background technology
Bacterial endotoxin is the lipopolysaccharides composition in the gram-negative bacterial cell wall structure, has multiple biologically active, is a kind of main pyrogen material.On medical industry, " pyrogen is exactly an endotoxin, and the control endogenous toxic material pollutes has just controlled pyrogen " become common recognition.The important of drug for injection checks that the pyrogen test of one of item is progressively replaced by bacterial endotoxin inspection.
The employed reagent of detection of bacterial endotoxin is tachypleus amebocyte lysate (TAL or LAL).TAL is a kind of biological reagent, contains factor C, factor B, proclotting enzyme and the coagulagen that can be activated by the denier bacterial endotoxin.(temperature, pH value and noiseless material etc.) under optimum conditions, bacterial endotoxin can make TAL produce agglutinating reaction, thereby produce gel.
The bacterial endotoxins test regulation that Chinese Pharmacopoeia is 2000 editions, get 10mm * 75mm test tube that 0.1ml tachypleus amebocyte lysate solution is housed, add 0.1ml need testing solution or standard endotoxin solution, put behind the mixing gently in 37 ± 1 ℃ of thermostats of people, be incubated 60 minutes ± 2 minutes, test tube is taken out from thermostat gently, slowly reversing is 180 °, and the pipe inner gel is indeformable, is not designated as the positive (+) from tube wall slippage person, gel can not be kept perfectly and from tube wall slippage person, be designated as feminine gender (-).The bacterial endotoxins test of Here it is conventional gel method.
In process of production, often will detect its bacteria endotoxin content, and whether decision can carry out subsequent processing production according to testing result to the semi-manufacture that come out from certain operation.Therefore, shorten detection time, promptly the detection of bacterial endotoxin method seems very important fast, and the isothermal reaction that conventional gel method detects need spend 60 minutes time, can not satisfy the demand of production practices.
Summary of the invention
The objective of the invention is to spend the long time in order to overcome existing detection of bacterial endotoxin method, the shortcomings such as demand that can not satisfy production practices provide a kind of and can shorten the reaction time, set up the method for fast measuring bacterial endotoxin, and prepare the quick detection kit of a kind of new bacterial endotoxin according to this method.
The preparation method of kit of the present invention is as follows:
(1) the sensitivity λ of selected fast detecting tachypleus amebocyte lysate
o
(2) tire the dilution standard endotoxin to a series of concentration by sign: 2 λ
o, λ
o, 0.5 λ
o, 0.25 λ
o
(3) with highly sensitive in λ
oConventional tachypleus amebocyte lysate and step (2) in the standard endotoxin series of the dilution mode and the conditioned response of pressing bacterial endotoxins test;
(4) determine that according to reaction result working as this routine tachypleus amebocyte lysate is λ by sensitivity
oT detection time during use
o
(5) get fast detecting tachypleus amebocyte lysate, standard endotoxin and bacterial endotoxin and check some of waters, pack in the packing box, detect the sign sensitivity λ of tachypleus amebocyte lysate in the lucid and lively speed of packing box subscript according to the convenient quantity collocation of using
oAnd reaction time T
oEvery batch of fast detecting tachypleus amebocyte lysate can indicate a plurality of sensitivity and corresponding reaction time simultaneously.
Described bacterial endotoxins test is determined reaction time T for the gel experimental method
o:
(1) routinely gel method to prepare concentration be λ
oOr λ
O1, λ
O2... the standard endotoxin solution;
(2) be λ with sensitivity, λ＜λ
o, λ
O1, λ
O2... conventional tachypleus amebocyte lysate and the standard endotoxin solution of step (1) preparation routinely gel method react, it is parallel that many groups are established in the endotoxic reaction of each concentration standard, when reaction proceeds to T
1, T
2, T
3... the time, described T
1, T
2, T
3...＜60 minutes, get one parallel group of gel method observing response result and record routinely respectively, when reaction when positive findings occurring the earliest, this time value is reaction time T that should the standard endotoxin concns
o
Described bacterial endotoxins test is determined reaction time T for the dynamic experiment method
o
(1) selected is λ (λ＜λ with sensitivity
o, λ
O1, λ
O2...) conventional tachypleus amebocyte lysate make the fast detecting tachypleus amebocyte lysate;
(2) routinely gel method to prepare concentration be 0.5 λ, λ, λ
o(or λ
O1, λ
O2...) the standard endotoxin solution;
(3) on the dynamic response instrument, react termination in 60 minutes with the tachypleus amebocyte lysate of step (1) and the standard endotoxin solution of step (2) preparation; If peaked 1/2 of transmittance (or the OD value) changing value of reaction performance graph when reaction terminating is gel thread, then λ
o(or λ
O1, λ
O2...) performance graph of concentration pairing shortest time of line segment of being in gel thread following (if OD value, then more than) is T
o
The present invention can improve detection efficiency, but the antijamming capability of tachypleus amebocyte lysate is not had influence.In Fig. 2, curve 7 and 13 is respectively and has added 0.5Eu/ml, the endotoxic test sample of 0.25Eu/ml standard and λ
oBe 0.25Eu/ml, T
oIt is 25 minutes the dynamic response curve of fast detecting TAL reaction of 25 clocks; Curve 16 and 21 is respectively and has added 60 minutes the dynamic response curve of general T AL reaction that 0.5Eu/ml, the endotoxic test sample of 0.25Eu/ml standard and sensitivity are 0.25Eu/ml.Can find out λ from these dynamic response curves
oBe 0.25Eu/ml, T
o25 minutes the result of fast detecting TAL reaction who is 25 minutes is that 60 minutes the result of general T AL reaction of 0.25Eu/ml is on all four with sensitivity.Adopt new method of the present invention, can detect conventional gel method needs 60 minute reaction time to foreshorten to 20 or 25 minutes, has improved the efficient that detects greatly, can satisfy the needs that the production run bacterial endotoxin detects fast.
The present invention will detect required tachypleus amebocyte lysate, and standard endotoxin and detection water are packaged into box, the convenient use.
Description of drawings
Fig. 1 is the dynamic response curve of standard endotoxin reaction of λ=0.06Eu/ml tachypleus amebocyte lysate and variable concentrations and gel figure as a result;
Fig. 2 is variable concentrations test sample positive control and the conventional tachypleus amebocyte lysate of identical sensitivity and the dynamic curve diagram of fast detecting tachypleus amebocyte lysate reaction;
Fig. 3 is a quick detection kit structural drawing of the present invention.
Embodiment
The principle of the quick detection kit of bacterial endotoxin is as follows:
The reaction of tachypleus amebocyte lysate and bacterial endotoxin has three important parameters: the activity of tachypleus amebocyte lysate, endotoxin concns and reaction time.To indicate sensitivity (λ) expression, activity is high more usually for the activity of tachypleus amebocyte lysate, and the λ value is more little.Conventional gel method is that the reaction time (T) was fixed as 60 minutes, with this understanding, the activity of tachypleus amebocyte lysate high more (the λ value is more little), detectable endotoxin concns is just low more.
The present invention improves endotoxin concns, and with this understanding, the tachypleus amebocyte lysate activity is high more, and the reaction time is just short more.According to this principle, use conventional high activity tachypleus amebocyte lysate (λ≤0.06Eu/ml), it is denoted as low activity λ
oUse (λ
oλ), promptly bring up to λ detecting endotoxin concns
o, make reaction time T
oReaction time T (60 minutes) less than routine.Realize this method, key is at selected λ
oAfterwards, how to come to determine corresponding reaction time T
o
Determine T
oTwo kinds of methods are arranged:
Method one: the gel experimental method is selected λ
o, λ for example
o=0.25Eu/ml; Dilution standard endotoxin to 2 λ
o, λ
o, 0.5 λ
oAnd 0.25 λ
oSeries concentration; (the high activity tachypleus amebocyte lysate of λ≤0.06Eu/ml) is reacted with this standard endotoxin serial dilutions to get conventional sign sensitivity and be λ.It is parallel that many groups are established in reaction.When reaction proceeds to T
1, T
2, T
3... the time (T
1, T
2, T
3...＜60 minutes), get one parallel group of observing response result respectively.The pairing reaction time is T when following reaction result occurring
o
Standard endotoxin concns (Eu/ml): 2 λ
oλ
o0.5 λ
o0.25 λ
o
Gel state: ++ ++ ++ ++--------
Method two: the dynamic experiment method get a conventional high activity tachypleus amebocyte lysate (λ≤0.06Eu/ml), with concentration be 0.5,0.25,0.125,0.06, the standard endotoxin solution of 0.03Eu/ml reacts on the dynamic response instrument, dynamic response curve and gel the results are shown in Figure 1.Can see that when being reacted to termination time T (60 minutes), the maximum changing value of transmittance is about 40% (100%-60%).For each response curve, if its transmittance changing value is greater than 1/2 maximum changing value (being that response curve terminates in below 80%), its gel state positive (+) is 0.5,0.25,0.125 and the response curve of 0.06Eu/ml as concentration; Otherwise gel state negative (-) is the response curve of 0.03Eu/ml as concentration.According to this rule, can in Fig. 1, find out selected λ easily
oPairing T
oValue.As selected λ
o=0.5Eu/ml, T
oBe the T among Fig. 1
O.5As selected λ
o=0.25Eu/ml, T
oBe the T among Fig. 1
O.25, or the like.With the checking of gel experimental method, also obtain identical experimental result (seeing Table 1).
The gel experimental method result of table 1 λ=0.06Eu/ml tachypleus amebocyte lysate
Embodiment:
1, test material
Standard endotoxin (RSE or CSE), tachypleus amebocyte lysate (TAL), bacterial endotoxin is checked water (W), apyrogeneity appliance one cover, one of vortex mixer, one of thermostat;
2, the preparation of the quick detection kit of bacterial endotoxin
(1) the sign sensitivity λ of the selected quick detection kit of bacterial endotoxin
o, as selecting preparation λ
oThe quick detection kit of=0.25Eu/ml.Same quick detection kit can have more than one to indicate sensitivity, as λ
O1=0.25Eu/ml, λ
O2=0.5Eu/ml or the like;
(2) determine the reaction time T of fast detecting tachypleus amebocyte lysate
o
The tachypleus amebocyte lysate of 1. getting sensitivity and be λ is some, requires λ＜λ
o, λ≤0.25 λ preferably
o, as λ
oDuring=0.25Eu/ml, λ≤0.06Eu/ml preferably.
2. get standard endotoxin (RSE or CSE) (bottle) and open, check that with bacterial endotoxin water is prepared into λ with it
oConcentration.Requiring of preparation method and conventional gel method is identical.If kit has a plurality of sign sensitivity, then need to prepare corresponding a plurality of concentration standard endotoxin solution;
3. get step tachypleus amebocyte lysate and the standard endotoxin solution reaction that 2. prepares of step 1., except that the reaction time has the requirement in addition, the method for reaction and condition are identical with conventional gel method requirement.Reaction is established some parallel group.
4. work as the parallel group reaction of 3. each of step and proceed to T respectively
1, T
2, T
3... the time (T
1, T
2, T
3...＜60 minutes), as 10,15,20,25...... minute, one by one reaction tube is taken out gel method requirement routinely in chronological order and observe gel state and write down the positive (+) or negative (-) as a result:
5. reaction result is the positive and time that the reaction time is the shortest is defined as T
oAs the test figure in the table 2, λ
o=0.5Eu/ml, T
o=25 minutes;
Table 2
| React parallel group | 1 2 3 4 5 6 7…… |
| Reaction time (minute) | 10 15 20 25 30 35 40…… |
| Gel state | ---- ---- ---- ++++ ++++ ++++ ++++…… |
6. different sign sensitivity has corresponding different T
oValue.As λ
O1=0.25Eu/ml, T
O1=25 minutes; λ
O2=0.5Eu/ml, T
O2=20 minutes ...;
(3) the assembling bacterial endotoxin detects box fast
The structure of the quick detection kit of bacterial endotoxin such as Fig. 3.Put 1 some of fast detecting tachypleus amebocyte lysate in the kit 4, some of standard endotoxins 2 (CSE), bacterial endotoxin is checked 3 some of waters, one of operation instructions.The box face is indicated the lot number of fast detecting tachypleus amebocyte lysate, sensitivity (λ
o), reaction time (T
o) and matters needing attention.
3, the using method of the quick detection kit of bacterial endotoxin
(1) selects the quick detection kit of suitable sensitivity as required;
(2) condition of gel method and requirement are used tachypleus amebocyte lysate, the standard endotoxin in the kit and are checked water routinely, and test sample is carried out detection of bacterial endotoxin, and unique difference is the isothermal reaction time only to need T
oMinute (T
o＜60).
Claims (3)
1. the preparation method of the quick detection kit of bacterial endotoxin, its step is as follows:
(1) the sensitivity λ of selected fast detecting tachypleus amebocyte lysate
O
(2) tire the dilution standard endotoxin to a series of concentration by sign: 2 λ
O, λ
O, 0.5 λ
O, 0.25 λ
O
(3) with highly sensitive in λ
OConventional tachypleus amebocyte lysate and step (2) in the standard endotoxin series of the dilution mode and the conditioned response of pressing bacterial endotoxins test;
(4) determine that according to reaction result working as this routine tachypleus amebocyte lysate is λ by sensitivity
OT detection time during use
O
(5) get fast detecting tachypleus amebocyte lysate, standard endotoxin and bacterial endotoxin and check some of waters, pack in the packing box, detect the sign sensitivity λ of tachypleus amebocyte lysate in the lucid and lively speed of packing box subscript according to the convenient quantity collocation of using
OAnd reaction time T
OEvery batch of fast detecting tachypleus amebocyte lysate can indicate a plurality of sensitivity and corresponding reaction time simultaneously.
2. according to the preparation method of the quick detection kit of the described bacterial endotoxin of claim 1, it is characterized in that: described bacterial endotoxins test is determined reaction time T for the gel experimental method
O:
(1) routinely gel method to prepare concentration be λ
OOr λ
O1, λ
O2The standard endotoxin solution;
(2) be λ with sensitivity, λ＜λ
O, λ
O1, λ
O2Conventional tachypleus amebocyte lysate and the standard endotoxin solution of step (1) preparation routinely gel method react, it is parallel that many groups are established in the endotoxic reaction of each concentration standard, when reaction proceeds to T
1, T
2, T
3The time, described T
1, T
2, T
3＜60 minutes, get one parallel group of gel method observing response result and record routinely respectively, when reaction when positive findings occurring the earliest, this time value is reaction time T that should the standard endotoxin concns
O
3. according to the preparation method of the quick detection kit of the described bacterial endotoxin of claim 1, it is characterized in that: described bacterial endotoxins test is determined reaction time T for the dynamic experiment method
O
(1) selected is λ with sensitivity, λ＜λ
O, λ
O1, λ
O2Conventional tachypleus amebocyte lysate make the fast detecting tachypleus amebocyte lysate;
(2) routinely gel method to prepare concentration be 0.5 λ, λ, λ
OOr λ
O1, λ
O2The standard endotoxin solution;
(3) on the dynamic response instrument, react termination in 60 minutes with the tachypleus amebocyte lysate of step (1) and the standard endotoxin solution of step (2) preparation; If the reaction performance graph when reaction terminating transmittance or OD value changing value peaked 1/2 be gel thread, then λ
OOr λ
O1, λ
O2The performance graph of concentration is in below the gel thread, if the OD value then more than, the pairing shortest time of line segment be T
O
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410028071 CN1721853B (en) | 2004-07-12 | 2004-07-12 | Preparation method of bacterial endotoxins rapid detecting reagent case |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410028071 CN1721853B (en) | 2004-07-12 | 2004-07-12 | Preparation method of bacterial endotoxins rapid detecting reagent case |
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| Publication Number | Publication Date |
|---|---|
| CN1721853A CN1721853A (en) | 2006-01-18 |
| CN1721853B true CN1721853B (en) | 2011-04-20 |
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ID=35912320
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410028071 Expired - Lifetime CN1721853B (en) | 2004-07-12 | 2004-07-12 | Preparation method of bacterial endotoxins rapid detecting reagent case |
Country Status (1)
| Country | Link |
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| CN (1) | CN1721853B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103675051B (en) * | 2013-12-20 | 2015-12-30 | 中国科学院苏州生物医学工程技术研究所 | A kind of method of endotoxin content in tracer liquid |
| CN104062163A (en) * | 2014-07-17 | 2014-09-24 | 厦门市鲎试剂实验厂有限公司 | Plasma endotoxin detection kit quality control product and preparation method thereof |
| CN109387638A (en) * | 2018-10-12 | 2019-02-26 | 四川升和药业股份有限公司 | Shenmai injection Test for Bacterial Endotoxins |
| CN109613228A (en) * | 2018-12-20 | 2019-04-12 | 湛江安度斯生物有限公司 | A method of reducing or eliminating aluminum hydroxide adjuvant interference detection of bacterial endotoxin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3149117C2 (en) * | 1981-12-11 | 1988-06-23 | Sartorius Gmbh, 3400 Goettingen, De | |
| CN1469124A (en) * | 2003-06-04 | 2004-01-21 | 湛江安度斯生物有限公司 | Homologous system model method for quantitatively detecting bacterial endotoxin of blood |
| CN1472333A (en) * | 2002-08-01 | 2004-02-04 | 辽宁味邦生物制药有限公司 | Inspection of endotoxin of collagenase cell by limulus reagent |
-
2004
- 2004-07-12 CN CN 200410028071 patent/CN1721853B/en not_active Expired - Lifetime
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3149117C2 (en) * | 1981-12-11 | 1988-06-23 | Sartorius Gmbh, 3400 Goettingen, De | |
| CN1472333A (en) * | 2002-08-01 | 2004-02-04 | 辽宁味邦生物制药有限公司 | Inspection of endotoxin of collagenase cell by limulus reagent |
| CN1469124A (en) * | 2003-06-04 | 2004-01-21 | 湛江安度斯生物有限公司 | Homologous system model method for quantitatively detecting bacterial endotoxin of blood |
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| Publication number | Publication date |
|---|---|
| CN1721853A (en) | 2006-01-18 |
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