CN108956967A - A kind of diluted plasma liquid kit and its application method for bacterial endotoxin gel method detection blood plasma - Google Patents
A kind of diluted plasma liquid kit and its application method for bacterial endotoxin gel method detection blood plasma Download PDFInfo
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- CN108956967A CN108956967A CN201810512354.2A CN201810512354A CN108956967A CN 108956967 A CN108956967 A CN 108956967A CN 201810512354 A CN201810512354 A CN 201810512354A CN 108956967 A CN108956967 A CN 108956967A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
A kind of diluted plasma liquid kit and its application method for bacterial endotoxin gel method detection blood plasma, the present invention relates to a kind of diluted plasma liquid kits and its application method for bacterial endotoxin gel method detection blood plasma, the purpose of the present invention is to solve gel methods to influence vulnerable to albumen in the anti-coagulants such as heparin sodium, sodium citrate in blood plasma and blood plasma, nephelometry is complicated for operation, and the problem of equipment valuableness.It include diluent A, dilution B and dilution C in kit of the present invention.It is diluted the blood plasma of different anti-coagulants using different diluent, targetedly eliminates interference of the anti-coagulants to reagents gel reaction, and can remove albumen in blood plasma, prevents the interference of albumen.And extension rate is smaller, diluting 8 times of conventional sensitivity of the limulus reagent can be detected.The present invention is applied to plasma endotoxin detection field.
Description
Technical field
The present invention relates to a kind of diluted plasma liquid kit for bacterial endotoxin gel method detection blood plasma and its make
Use method.
Background technique
Biological products containing plasma composition, including platelet lysates liquid, platelet rich plasma, normal plasma etc. orthopaedics,
The departments such as dermatology, ophthalmology are widely used.If bacterial endotoxin pollutes this based article, body can be caused to feel cold after
Fever, leucocyte decline, vasopermeability increase etc. " heat source qualitative response ", therefore plasma endotoxin content detection in clinic and is returned
It is of great significance in defeated application.Gel method and nephelometry, turbidity are defined as in the method pharmacopeia of detection plasma endotoxin at present
Method although specific high sensitivity the features such as, but operate increasingly complex, and need extra purchase expensive device.Gel method operation letter
Single, applicability is preferable, but influences vulnerable to albumen in the anti-coagulants such as heparin sodium, sodium citrate in blood plasma and blood plasma, produces to result
Raw interference.It is also different to the interference principle of reagents because heparin sodium is different from the anticoagulant principle of sodium citrate.Although larger multiple
Dilution can eliminate interference, but also to the sensitivity of reagents, more stringent requirements are proposed simultaneously.Laboratory is unified mostly at present
It uses water as the dilution of the tested substance containing plasma composition using endotoxin detection, corresponding anti-interference work cannot be played
With.In addition interim existing with corresponding anti-interference dilution process, since the concentration of the not perfect and anti-interference substance of preparing process is matched
System inaccuracy, also cannot achieve the Sensitive Detection of reagents.
Summary of the invention
The purpose of the present invention is to solve gel methods vulnerable to the anti-coagulants such as heparin sodium, sodium citrate and blood plasma in blood plasma
Middle albumen influences, nephelometry is complicated for operation, and the problem of equipment valuableness, and the present invention provides one kind to be used for bacterial endotoxin gel
The diluted plasma liquid kit and its application method of method detection blood plasma.
A kind of diluted plasma liquid kit for bacterial endotoxin gel method detection blood plasma of the present invention includes dilution
A, dilution B and dilution C；Wherein the diluent A is the protamine sulfate solution that concentration is 0.1-0.3mg/mL；It is dilute
Releasing liquid B is the CaCl that concentration is 8.0-10.0mg/mL2Solution；Dilution C is made of Hepes and Triton X-100, dilution
The final concentration of 8-10mmol/L of Hepes in C, the final concentration of 0.3%-0.5% of Triton X-100.
A kind of application method of the diluted plasma liquid kit for bacterial endotoxin gel method detection blood plasma of the present invention
Are as follows: test plasma and diluent A or dilution B are mixed, 2-4min is then allowed to stand, dilution C is then added, is stood after mixing
4-6min obtains test sample, and test sample is added separately in test sample pipe and test sample positive pipe, 37 DEG C of water-bath 1h；It is negative
Pipe is feminine gender；Positive pipe and test sample positive pipe are the positive, then test establishment；If wherein test plasma anti-coagulants is heparin class
Anti-coagulants then selects diluent A, if blood anticoagulant to be measured is sodium citrate class anti-coagulants, selects dilution B.
Beneficial effects of the present invention:
Either sodium citrate anticoagulant or sodium heparin anticoagulant, kit of the invention can use lesser dilution times
Number relatively sensitively detects the endotoxin contained in blood plasma, and can effectively eliminate the dry of blood plasma induced by endotoxin reagents reaction
It disturbs.
The present invention is different for the action principle of different anti-coagulants and different reagents is used to carry out anti-interference dilution, specific aim
By force, effect is good, and extension rate is small, only dilutes 8 times, and entire experimental implementation is simple, without adding other ancillary equipments,
Without changing experiment equipment and equipment, cost is lower, and the effect of manufactured goods is more preferable, is conducive to the work quality of medical testing agency
With the raising of working efficiency.
Specific embodiment
Technical solution of the present invention is not limited to the specific embodiment of act set forth below, further include each specific embodiment it
Between any combination.
Specific embodiment 1: a kind of diluted plasma for bacterial endotoxin gel method detection blood plasma of present embodiment
Liquid kit includes diluent A, dilution B and dilution C；It is 0.1-0.3mg/mL's that wherein the diluent A, which is concentration,
Protamine sulfate solution；Dilution B is the CaCl that concentration is 8.0-10.0mg/mL2Solution；Dilution C by Hepes with
Triton X-100 is formed, and the final concentration of 8-10mmol/L of Hepes in dilution C, Triton X-100's is final concentration of
0.3%-0.5%.
Present embodiment the utility model has the advantages that
Present embodiment is different for the action principle of different anti-coagulants and different reagents is used to carry out anti-interference dilution, needle
Strong to property, effect is good, and extension rate is small, only dilutes 8 times, entire experimental implementation is simple, without adding other auxiliary
Equipment, at low cost without changing experiment equipment and equipment, the effect of manufactured goods is good, is conducive to the as received basis of medical testing agency
The raising of amount and working efficiency.
Specific embodiment 2: the present embodiment is different from the first embodiment in that: diluent A is that concentration is
The protamine sulfate solution of 0.2mg/mL.Other are the same as one or two specific embodiments.
Specific embodiment 3: the present embodiment is different from the first and the second embodiment in that: dilution B is that concentration is
The CaCl of 9.0mg/mL2Solution.It is the same as one or two specific embodiments.
Specific embodiment 4: unlike one of present embodiment and specific embodiment one to three: in dilution C
The final concentration of 9mmol/L of Hepes.Other are identical as one of specific embodiment one to three.
Specific embodiment 5: unlike one of present embodiment and specific embodiment one to four: in dilution C
Final concentration of the 0.4% of Triton X-100.Other are identical as one of specific embodiment one to four.
Specific embodiment 6: a kind of diluted plasma for bacterial endotoxin gel method detection blood plasma of present embodiment
The application method of liquid kit are as follows: test plasma and diluent A or dilution B are mixed, 2-4min is then allowed to stand, is then added
Dilution C stands 4-6min after mixing, obtains test sample, and test sample is added separately to test sample pipe and test sample positive pipe
In, 37 DEG C of water-bath 1h；Negative tube is feminine gender；Positive pipe and test sample positive pipe are the positive, then test establishment；If wherein to be measured
Plasma anticoagulant agent is heparin class anti-coagulants, then selects diluent A, if blood anticoagulant to be measured is sodium citrate class anti-coagulants,
Select dilution B.
Specific embodiment 7: present embodiment is unlike specific embodiment six: test plasma and diluent A or
1:1 is mixed dilution B by volume.Other are identical as specific embodiment six.
Specific embodiment 8: present embodiment present embodiment is unlike specific embodiment six or seven: to be measured
1:6 is mixed blood plasma by volume with dilution C.Other are identical as specific embodiment six or seven.
Beneficial effects of the present invention are verified using following embodiment:
Embodiment one: a kind of to detect the diluted plasma liquid kit of blood plasma comprising diluting for bacterial endotoxin gel method
Liquid A, dilution B and dilution C；Wherein the diluent A is the protamine sulfate solution that concentration is 0.1-0.3mg/mL；
Dilution B is the CaCl that concentration is 8.0-10.0mg/mL2Solution；Dilution C is made of Hepes and Triton X-100, dilution
The final concentration of 8-10mmol/L of Hepes in liquid C, the final concentration of Triton X-100 is in 0.3%-0.5%.
A kind of user of the diluted plasma liquid kit for bacterial endotoxin gel method detection blood plasma of the present embodiment
Method are as follows: test plasma and diluent A or dilution B are mixed, 3min is then allowed to stand, dilution C is then added, is stood after mixing
5min obtains test sample, and test sample is added separately in test sample pipe and test sample positive pipe, 37 DEG C of water-bath 1h；If wherein
Test plasma anti-coagulants is heparin class anti-coagulants, then selects diluent A, test tube is gently taken out from water-bath, is slowly reversed
At 180 DEG C, pipe inner gel is indeformable, is not the positive from tube wall slippage person, is recorded as (+)；Gel is not able to maintain complete and from pipe
Wall slippage person is feminine gender, is recorded as (-).Negative tube be feminine gender, the positive pipe and test sample positive pipe be the positive, then experiment at
It is vertical.
Endotoxin detection is carried out to the former blood plasma using sodium citrate anticoagulant preparation, verifying the present embodiment kit has
Effect property, while carrying out the comparative experiments that method exclusive PCR is diluted with water in sample baterial endotoxin test.
1. acquiring peripheral blood 80ml with 200ml blood taking bag (including sodium citrate 28ml).80ml volume is free of anti-coagulants.
2. being centrifugated out blood plasma 56ml.
3. taking three empty ampoule bottles, label 1,2,3 is separately added into 0.9ml blood plasma.The endogenous toxic material of 10EU/ml is added to headpin
Plain working standard 0.1ml, the final concentration of 1EU/ml of endotoxin in blood plasma；The endotoxin work of 20EU/ml is added into No. 2 bottles
Standard items 0.1ml, the final concentration of 2EU/ml of endotoxin in blood plasma；Add the endotoxin working standard of 40EU/ml into No. 3 bottles
0.1ml, the final concentration of 4EU/ml of endotoxin in blood plasma are mixed spare.
4. carrying out endotoxin detection to 1,2, No. 3 bottle sample respectively using this kit method, the reagents used is sensitive
Degree is 0.25EU/ml.
5. method, which is diluted with water, using baterial endotoxin test carries out endotoxin detection, extension rate to 1,2, No. 3 bottle sample
For 10 times, 40 times, 100 times, the sensitivity of the limulus reagent used is 0.25EU/ml.
6. data result
1 RNA isolation kit of table
2 dilution method of table
Kit method to Sample Dilution multiple be 8 times, 1 after dilution, 2, No. 3 sample endotoxin contents be respectively
0.125EU/ml, 0.25EU/ml, 0.5EU/ml, theoretically in addition to No. 1 sample, 2, No. 3 sample standard deviations reach the spirit of reagents reaction
Sensitivity value should all detect, from table 1 it follows that can to detect endotoxin in 2, No. 3 samples exceeded for kit method, and supply
The test product positive is set up, and illustrates that kit method can effectively eliminate the interference of blood plasma induced by endotoxin reaction, and more sensitive.
Comparison dilution method: method is diluted with water using baterial endotoxin test, 10,40,100 is diluted respectively to 1,2, No. 3 sample
Times, endotoxin content is 0.4EU/ml after No. 3 10 times of Sample Dilution, and theory should detect, remaining all sample endotoxin content is equal
Not up to sensitivity of the limulus reagent value can not detect, from Table 2, it can be seen that all samples 10,40 times dilution under, to horseshoe crab
Reagent reaction still has interference, and the lower interference of 100 times of dilutions is eliminated.No. 3 samples interfere under 10 times of dilutions because sample exists, and do not examine
Out.
Endotoxin detection is carried out to the former blood plasma using sodium heparin anticoagulant preparation, verifies the validity of this kit.Together
The comparative experiments of method exclusive PCR is diluted with water in Shi Jinhang sample baterial endotoxin test.
1. acquiring peripheral blood 10ml with 10ml heparin sodium heparin tube.
2. being centrifugated out blood plasma 4ml.
3. taking three empty ampoule bottles, label 1,2,3 is separately added into 0.9ml blood plasma.The endogenous toxic material of 10EU/ml is added to headpin
Plain working standard 0.1ml, the final concentration of 1EU/ml of endotoxin in blood plasma；The endotoxin work of 20EU/ml is added into No. 2 bottles
Standard items 0.1ml, the final concentration of 2EU/ml of endotoxin in blood plasma；Add the endotoxin working standard of 40EU/ml into No. 3 bottles
0.1ml, the final concentration of 4EU/ml of endotoxin in blood plasma are mixed spare.
4. carrying out endotoxin detection to 1,2, No. 3 bottle sample respectively using this kit method, the reagents used is sensitive
Degree is 0.25EU/ml.
5. method, which is diluted with water, using baterial endotoxin test carries out endotoxin detection, extension rate to 1,2, No. 3 bottle sample
For 10 times, 40 times, 100 times, the sensitivity of the limulus reagent used is 0.25EU/ml.
6. data result
3 RNA isolation kit of table
4 dilution method of table
Kit method to Sample Dilution multiple be 8 times, 1 after dilution, 2, No. 3 sample endotoxin contents be respectively
0.125EU/ml, 0.25EU/ml, 0.5EU/ml, theoretically in addition to No. 1 sample, 2, No. 3 sample standard deviations reach the spirit of reagents reaction
Sensitivity value should all detect, from table 3 it is observed that can to detect endotoxin in 2, No. 3 samples exceeded for kit method, and supply
The test product positive is set up, and illustrates that kit method can effectively eliminate the interference of blood plasma induced by endotoxin reaction, and more sensitive.
Dilution method: being diluted with water method using baterial endotoxin test and dilute 10,40,100 times respectively to 1,2, No. 3 sample, and 3
Endotoxin content is 0.4EU/ml after numbers 10 times of Sample Dilution, and theory should detect, remaining all sample endotoxin content does not reach
To sensitivity of the limulus reagent value, can not detect, from Table 2, it can be seen that all samples 10,40 times dilution under, to reagents
Reaction still has interference, and the lower interference of 100 times of dilutions is eliminated.No. 3 samples interfere under 10 times of dilutions because sample exists, and are not detected.
Therefore, either sodium citrate anticoagulant or sodium heparin anticoagulant, the present embodiment RNA isolation kit can be used smaller
Extension rate more sensitively detect the endotoxin contained in blood plasma, and it is anti-to effectively eliminate blood plasma induced by endotoxin reagents
The interference answered.
Claims (8)
1. a kind of diluted plasma liquid kit for bacterial endotoxin gel method detection blood plasma, it is characterised in that the kit
Include diluent A, dilution B and dilution C；Wherein the diluent A is the sulfuric acid milt that concentration is 0.1-0.3mg/mL
Protein solution；Dilution B is the CaCl that concentration is 8.0-10.0mg/mL2Solution；Dilution C is by Hepes and Triton X-100
It forms, the final concentration of 8-10mmol/L, the final concentration of 0.3%-0.5% of Triton X-100 of Hepes in dilution C.
2. a kind of plasma extender reagent for bacterial endotoxin gel method detection blood plasma according to claim 1
Box, it is characterised in that diluent A is the protamine sulfate solution that concentration is 0.2mg/mL.
3. a kind of plasma extender reagent for bacterial endotoxin gel method detection blood plasma according to claim 1
Box, it is characterised in that dilution B is the CaCl that concentration is 9.0mg/mL2Solution.
4. a kind of plasma extender reagent for bacterial endotoxin gel method detection blood plasma according to claim 1
Box, it is characterised in that the final concentration of 9mmol/L of Hepes in dilution C.
5. a kind of plasma extender reagent for bacterial endotoxin gel method detection blood plasma according to claim 1
Box, it is characterised in that final concentration of the 0.4% of Triton X-100 in dilution C.
6. a kind of diluted plasma liquid kit for bacterial endotoxin gel method detection blood plasma as described in claim 1
Application method, it is characterised in that application method are as follows: test plasma and diluent A or dilution B are mixed, 2- is then allowed to stand
4min, then be added dilution C, stand 4-6min after mixing, obtain test sample, by test sample be added separately to test sample pipe and
In test sample positive pipe, 37 DEG C of water-bath 1h；Negative tube is feminine gender；The positive pipe and test sample positive pipe be the positive, then experiment at
It is vertical；If wherein test plasma anti-coagulants is heparin class anti-coagulants, diluent A is selected, if blood anticoagulant to be measured is citric acid
Sodium class anti-coagulants, then select dilution B.
7. a kind of diluted plasma liquid kit for bacterial endotoxin gel method detection blood plasma according to claim 6
Application method, it is characterised in that 1:1 is mixed by volume by test plasma and diluent A or dilution B.
8. a kind of diluted plasma liquid kit for bacterial endotoxin gel method detection blood plasma according to claim 6
Application method, it is characterised in that 1:6 is mixed test plasma by volume with dilution C.
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| CN1407900A (en) * | 1999-10-14 | 2003-04-02 | 人类基因组科学公司 | Methods of treating or preventing cell, tissue, and organ damage using human myeloid progenitor inhibitory factor-1 (MPIF-1) |
| CN102141570A (en) * | 2009-12-25 | 2011-08-03 | 希森美康株式会社 | Activated partial thromboplastin time measuring reagent, activated partial thromboplastin time measuring method, and determination method for determining presence or absence of blood coagulation inhibitor |
| CN202119777U (en) * | 2011-06-23 | 2012-01-18 | 厦门市鲎试剂实验厂有限公司 | Limulus kit for rapidly testing endotoxin |
| CN103063592A (en) * | 2012-12-31 | 2013-04-24 | 中国医学科学院输血研究所 | Determination method for heparin activity |
| CN104232739A (en) * | 2014-08-18 | 2014-12-24 | 中国大冢制药有限公司 | Method for detecting bacterial endotoxin in citric acid raw material |
| 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 |
| CN106645665A (en) * | 2016-12-27 | 2017-05-10 | 北京赛科希德科技股份有限公司 | Thrombin time detection reagent |
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2018
- 2018-05-24 CN CN201810512354.2A patent/CN108956967A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1407900A (en) * | 1999-10-14 | 2003-04-02 | 人类基因组科学公司 | Methods of treating or preventing cell, tissue, and organ damage using human myeloid progenitor inhibitory factor-1 (MPIF-1) |
| CN102141570A (en) * | 2009-12-25 | 2011-08-03 | 希森美康株式会社 | Activated partial thromboplastin time measuring reagent, activated partial thromboplastin time measuring method, and determination method for determining presence or absence of blood coagulation inhibitor |
| CN202119777U (en) * | 2011-06-23 | 2012-01-18 | 厦门市鲎试剂实验厂有限公司 | Limulus kit for rapidly testing endotoxin |
| CN103063592A (en) * | 2012-12-31 | 2013-04-24 | 中国医学科学院输血研究所 | Determination method for heparin activity |
| 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 |
| CN104232739A (en) * | 2014-08-18 | 2014-12-24 | 中国大冢制药有限公司 | Method for detecting bacterial endotoxin in citric acid raw material |
| CN106645665A (en) * | 2016-12-27 | 2017-05-10 | 北京赛科希德科技股份有限公司 | Thrombin time detection reagent |
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