CN109521204A - A kind of lupus anticoagulant detection reagent and preparation method thereof and application method - Google Patents
A kind of lupus anticoagulant detection reagent and preparation method thereof and application method Download PDFInfo
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Abstract
The invention belongs to technical field of biological; disclose a kind of lupus anticoagulant detection reagent and preparation method thereof and application method; the lupus anticoagulant detection reagent mainly includes screening agent, Confirmation reagent, complex reagent, and screening agent includes: that percentage by volume is the coagulation factor activator of 13mg/L, the buffer of 65mmol/LpH=7.5,55mg/L synthetic phospholipid, 75g/L protein protective agent, 0.06% preservative, 100mmol/L ionic strength adjustor and 8g/L coagulation factor protective agent；Confirm that agent synthetic phospholipid composition is higher.Reagent of the present invention is liquid reagent; preparation process is easy; advantage of lower cost; by the way that protein protective agent is added; the stability of liquid reagent is effectively ensured, effectively reduces false positive rate and false negative rate in testing result, by the way that different synthetic phospholipids are respectively adopted from Confirmation reagent to screening agent; the sensitivity to LA detection is improved, the accuracy of detection test is improved.
Description
Technical field
The invention belongs to biological detection reagent technical field more particularly to a kind of lupus anticoagulant detection reagent and its preparations
Method and application method.
Background technique
Currently, the prior art commonly used in the trade is such that
Lupus anticoagulant (Lupus anticoagulants, LA) is a kind of pathologic circulating anticoagulant that phosphatide relies on
Matter is the heterogeneous immunoglobulin of IgG or IgM, by combining 2-glycoprotein I, human thrombin original and other phosphatide
Compound makes cruor time extending come the coagulation process for interfering phosphatide to rely on.Lasting LA positive patient is considered having higher
Thrombosis and risk of recurrence and unknown cause habitual abortion, stillborn foetus, easy bolt disease and certain autoimmune diseases
Danger signal.The detection of LA includes Screening tests, validation test and composite testing.Due to the heterogeneity and detection examination of LA antibody
The diversity of agent, influence factor is many and diverse in detection process, and LA detection there is no reference material and with reference to method at present, therefore how
The accuracy for ensuring testing result is a major challenge for laboratory testing personnel.
The key for mostly using APTT reagent and DRVVT both methods to be used to detect lupus anticoagulant at present is to screen examination
Agent and the matching for making a definite diagnosis detection reagent, including activation ingredient and phospholipid composition used, since the RVV in DRVVT is from pallas pit viper
A kind of enzyme that venom extracts, stability is very poor in the solution.Therefore DRVVT reagent can only be made into freeze-dried powder reagent.Due to stabilization
Property the problem of, DRVVT reagent will be finished as early as possible after redissolution, and freeze-dried powder reagent needs to redissolve when in use, redissolve when due to
The difference of the redissolution dosage of addition can also cause very big difference.Therefore the screening reagent and freeze-dried powder form of freeze-dried powder form
The ingredient for making a definite diagnosis reagent is difficult to match, so that the false positive rate of testing result or false negative rate increase.
In conclusion problem of the existing technology is:
(1) currently without the reference material detected to LA and with reference to method, for ensuring the accuracy of testing result to reality
Testing staff is tested with biggish challenge.
(2) since the RVV in DRVVT is a kind of enzyme extracted from pit viper venom, stability is very poor in the solution, easily causes
False positive rate and false negative rate in testing result increase.
Solve the difficulty and meaning of above-mentioned technical problem: the present invention, can be for LA's by the improvement to DRVVT stability
Detection provides strong reference, substantially increases the accuracy of testing result.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of lupus anticoagulant detection reagents and preparation method thereof
And application method.
The invention is realized in this way a kind of lupus anticoagulant detection reagent mainly includes screening agent, Confirmation reagent, answers
Close reagent.
Screening agent include: percentage by volume be the coagulation factor activator of 13mg/L, 65mmol/L pH=7.5 it is slow
Fliud flushing, 55mg/L synthetic phospholipid, 75g/L protein protective agent, 0.06% preservative, 100mmol/L ionic strength adjustor and 8g/
L coagulation factor protective agent.
Confirmation agent includes: the buffering that percentage by volume is the coagulation factor activator of 13mg/L, 65mmol/L pH=7.5
Liquid, 120mg/L synthetic phospholipid, 75g/L protein protective agent, 0.06% preservative, 100mmol/L ionic strength adjustor and 8g/L
Coagulation factor protective agent.
Complex reagent includes: that patients blood plasma, human normal plasma are mixed by 1:1.
Further, the coagulation factor activator is silica；Buffer is trishydroxymethylaminomethane-hydrochloride buffer
Liquid.
Further, synthetic phospholipid is soybean lecithin in the screening agent, confirms that phosphatide is hydrogenated phospholipid in agent.
Further, the protein protective agent are as follows: BSA, casein or skimmed milk power, sucrose, amino acid, trehalose, sweet dew
The one or several kinds of alcohol；Preservative is Sodium azide；Ionic strength adjustor are as follows: NaCl or KCl；Coagulation factor protective agent are as follows:
Glycine.
Another object of the present invention is to provide a kind of preparation method of lupus anticoagulant detection reagent, specifically:
Screening agent:
Step 1: the Tris-HCl buffer solution of corresponding pH value is configured by a certain percentage；
Step 2: it weighs 5mg soybean lecithin and 5mlTris-HCl buffer solution is added, be ground to emulsus, visually observe without bright
Aobvious particle, acquisition soybean lecithin Stock concentrations are 1g/L；
Step 3: a certain proportion of ionic strength adjustor is added into 100ml volumetric flask, coagulation factor activator, prevents
Rotten agent, protein protective agent, coagulation factor protective agent are and 0.5ml soybean lecithin stock solution that Tris-HCl solution is added to scale
Line is uniformly mixed, and is saved in 2~8 DEG C.
Confirm agent:
Step 1: the Tris-HCl buffer solution of corresponding pH value is configured by a certain percentage；
Step 2: it weighs 5mg hydrogenated phospholipid and 5mlTris-HCl buffer solution is added, be ground to emulsus, visually observe without bright
Aobvious particle, acquisition soybean lecithin Stock concentrations are 1g/L；
Step 3: a certain proportion of ionic strength adjustor is added into 100ml volumetric flask, coagulation factor activator, prevents
Rotten agent, protein protective agent, coagulation factor protective agent are and 0.5ml hydrogenated phospholipid stock solution that Tris-HCl solution is added to scale
Line is uniformly mixed, and is saved in 2~8 DEG C.
Another object of the present invention is to provide a kind of application method of lupus anticoagulant detection reagent, specifically:
Step 1: taking detection blood plasma to mix with screening agent, be put into coagulo meter, when obtaining the screening blood coagulation of detection blood sample
Between, testing result is obtained, if testing result is positive, continues Confirmation reagent detection；
Step 2: screening positive detection blood sample being mixed with Confirmation reagent, is put into coagulo meter, obtains detection blood sample really
Recognize the clotting time, obtain testing result, if result is still the positive, continues complex reagent detection；
Step 3: it will test after blood sample mixes with normal person blood sample by 1:1, while using screening agent and Confirmation reagent
It is detected, obtains the clotting time.
In conclusion advantages of the present invention and good effect are as follows: reagent of the present invention is liquid reagent, compared to freeze-drying
Pulvis preparation process is easy, advantage of lower cost, by the way that protein protective agent is added, the stability of liquid reagent is effectively ensured, has
Effect reduces false positive rate and false negative rate in testing result, different by the way that screening agent is respectively adopted from Confirmation reagent
Synthetic phospholipid improves the sensitivity to LA detection, improves the accuracy of detection test.
Detailed description of the invention
Fig. 1 is screening agent preparation flow figure provided in an embodiment of the present invention；
Fig. 2 is Confirmation reagent preparation flow figure provided in an embodiment of the present invention；
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Lupus anticoagulant detection reagent provided in an embodiment of the present invention mainly includes screening agent, Confirmation reagent, compound examination
Agent.
Screening agent provided in an embodiment of the present invention include: percentage by volume be 13mg/L coagulation factor activator,
Buffer, 55mg/L synthetic phospholipid, 75g/L protein protective agent, 0.06% preservative, 100mmol/ of 65mmol/L pH=7.5
L ionic strength adjustor and 8g/L coagulation factor protective agent.
It is provided in an embodiment of the present invention confirmation agent include: percentage by volume be 13mg/L coagulation factor activator,
The buffer of 65mmol/L pH=7.5,120mg/L synthetic phospholipid, 75g/L protein protective agent, 0.06% preservative,
100mmol/L ionic strength adjustor and 8g/L coagulation factor protective agent.
Complex reagent provided in an embodiment of the present invention includes: that patients blood plasma, human normal plasma are mixed by 1:1.
Coagulation factor activator provided in an embodiment of the present invention is silica；Buffer is trishydroxymethylaminomethane-salt
Acid buffer.
Synthetic phospholipid is soybean lecithin in screening agent provided in an embodiment of the present invention, confirms that phosphatide is hydrogenation phosphorus in agent
Rouge.
Protein protective agent provided in an embodiment of the present invention are as follows: BSA, casein or skimmed milk power, sucrose, amino acid, seaweed
Sugar, the one or several kinds of mannitol.
Preservative provided in an embodiment of the present invention is Sodium azide.
Ionic strength adjustor provided in an embodiment of the present invention are as follows: NaCl or KCl.
Coagulation factor protective agent provided in an embodiment of the present invention are as follows: glycine.
Application principle of the invention is described in detail with reference to the accompanying drawing.
As shown in Figure 1, a kind of preparation method for detecting lupus anticoagulant reagent provided in an embodiment of the present invention, specifically includes
Following steps:
S101: configuring the Tris-HCl buffer solution of corresponding pH value by a certain percentage, spare；
S102: it prepares soybean lecithin stock solution: weighing 5mg soybean lecithin and 5mlTris-HCl buffer solution is added, using equal
Matter machine carries out average value processing, and acquisition soybean lecithin Stock concentrations are 1g/L；
S103: it prepares hydrogenated phospholipid stock solution: weighing 5mg hydrogenated phospholipid and 5mlTris-HCl buffer solution is added, using equal
Matter machine carries out average value processing, and acquisition hydrogenated phospholipid Stock concentrations are 1g/L；
S104: it prepares screening agent: a certain proportion of ionic strength adjustor, coagulation factor activation being added into volumetric flask
Agent, preservative, protein protective agent, coagulation factor protective agent are and 0.5ml soybean lecithin stock solution to be ultrasonically treated 5min, be added
Tris-HCl solution shakes up, saves backup in 2~8 DEG C to volumetric flask graduation mark；
S105: a certain proportion of ionic strength adjustor, coagulation factor activation preparation confirmation agent: are added into volumetric flask
Agent, preservative, protein protective agent, coagulation factor protective agent are and 0.5ml hydrogenated phospholipid stock solution to be ultrasonically treated 5min, be added
Tris-HCl solution shakes up, saves backup in 2~8 DEG C to graduation mark；
S106: prepare complex reagent: patients blood plasma is mixed with ordinary person's blood plasma by 1:1, is saved backup.
Ultrasonic treatment provided in an embodiment of the present invention is to carry out ultrasonic vibration, 25 DEG C, 300W to volumetric flask using ultrasonic wave
It is ultrasonically treated 5min.
Average value processing provided in an embodiment of the present invention is that average value processing is carried out using homogenizer to visually observing without obvious
Grain.
Volumetric flask specification provided in an embodiment of the present invention is 100mL.
As shown in Fig. 2, a kind of application method of lupus anticoagulant detection reagent provided in an embodiment of the present invention, specifically:
S201: taking detection blood plasma to mix with screening agent, be put into coagulo meter, when obtaining the screening blood coagulation of detection blood sample
Between, testing result is obtained, if testing result is positive, continues Confirmation reagent detection；
S202: screening positive detection blood sample being mixed with Confirmation reagent, is put into coagulo meter, obtains the confirmation of detection blood sample
Clotting time obtains testing result, if result is still the positive, continues complex reagent detection；
S203: will test after blood sample mixes with the blood sample of normal person by 1:1, at the same using screening agent and Confirmation reagent into
Row detection, obtains the clotting time.
The present invention is described further combined with specific embodiments below.
Embodiment 1
With kit prepared by the present invention and import other brands LA detection reagent respectively to clinical 200 plasma samples into
The screening test of row LA counts the extended incidence of plasma coagulation time, to investigate the clinical specificity of reagent, as a result such as table 1
It is shown.
Table 1
As can be seen from Table 1, the clinical specificity of kit of the present invention reaches import reagent standard.
Embodiment 2
6 DEG C of preservations are placed in kit prepared by the present invention, and when use takes out, and it is normal to measure same LA in different time
The setting time of blood plasma, LA abnormal plasma, the results are shown in Table 2.
Table 2
Such as table 2, as a result kit METHOD FOR CONTINUOUS DETERMINATION of the present invention 15 days is always maintained at stabilization, illustrates that kit of the present invention has
Preferable stability.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (6)
1. a kind of lupus anticoagulant detection reagent and preparation method thereof and application method, which is characterized in that the lupus anticoagulant
Detection reagent mainly includes screening agent, Confirmation reagent, complex reagent；
Screening agent include: percentage by volume be the coagulation factor activator of 13mg/L, 65mmol/L pH=7.5 buffer,
55mg/L synthetic phospholipid, 75g/L protein protective agent, 0.06% preservative, 100mmol/L ionic strength adjustor and 8g/L blood coagulation
Factor protective agent；
Confirmation agent include: percentage by volume be the coagulation factor activator of 13mg/L, 65mmol/L pH=7.5 buffer,
120mg/L synthetic phospholipid, 75g/L protein protective agent, 0.06% preservative, 100mmol/L ionic strength adjustor and 8g/L are solidifying
Blood factor protective agent；
Complex reagent includes: that patients blood plasma, human normal plasma are mixed by 1:1.
2. a kind of lupus anticoagulant detection reagent and preparation method thereof and application method as described in claim 1, which is characterized in that
The coagulation factor activator is silica；Buffer is tris-HCI buffer.
3. a kind of lupus anticoagulant detection reagent and preparation method thereof and application method as described in claim 1, which is characterized in that
Synthetic phospholipid is soybean lecithin in the screening agent, confirms that phosphatide is hydrogenated phospholipid in agent.
4. a kind of lupus anticoagulant detection reagent and preparation method thereof and application method as described in claim 1, which is characterized in that
The protein protective agent are as follows: BSA, casein or skimmed milk power, sucrose, amino acid, trehalose, mannitol it is a kind of or several
Kind；Preservative is Sodium azide；Ionic strength adjustor are as follows: NaCl or KCl；Coagulation factor protective agent are as follows: glycine.
5. a kind of lupus anticoagulant detection reagent and preparation method thereof and application method as described in claim 1, which is characterized in that
A kind of preparation method of the lupus anticoagulant detection reagent, specifically:
Screening agent:
Step 1: the Tris-HCl buffer solution of corresponding pH value is configured by a certain percentage；
Step 2: it weighs 5mg soybean lecithin and 5mlTris-HCl buffer solution is added, be ground to emulsus, visually observe without obvious
Grain, acquisition soybean lecithin Stock concentrations are 1g/L；
Step 3: be added into 100ml volumetric flask a certain proportion of ionic strength adjustor, coagulation factor activator, preservative,
Protein protective agent, coagulation factor protective agent are and 0.5ml soybean lecithin stock solution, addition Tris-HCl solution to graduation mark mix
It closes uniformly, is saved in 2~8 DEG C；
Confirm agent:
Step 1: the Tris-HCl buffer solution of corresponding pH value is configured by a certain percentage；
Step 2: it weighs 5mg hydrogenated phospholipid and 5mlTris-HCl buffer solution is added, be ground to emulsus, visually observe without obvious
Grain, acquisition soybean lecithin Stock concentrations are 1g/L；
Step 3: be added into 100ml volumetric flask a certain proportion of ionic strength adjustor, coagulation factor activator, preservative,
Protein protective agent, coagulation factor protective agent are and 0.5ml hydrogenated phospholipid stock solution, addition Tris-HCl solution to graduation mark mix
It closes uniformly, is saved in 2~8 DEG C.
6. a kind of lupus anticoagulant detection reagent and preparation method thereof and application method as described in claim 1, which is characterized in that
A kind of application method of the lupus anticoagulant detection reagent, specifically:
Step 1: taking detection blood plasma to mix with screening agent, be put into coagulo meter, obtain the screening clotting time of detection blood sample,
Testing result is obtained, if testing result is positive, continues Confirmation reagent detection；
Step 2: screening positive detection blood sample being mixed with Confirmation reagent, is put into coagulo meter, and the confirmation for obtaining detection blood sample is solidifying
The blood time obtains testing result, if result is still the positive, continues complex reagent detection；
Step 3: it will test after blood sample mixes with normal person blood sample by 1:1, while being carried out using screening agent and Confirmation reagent
Detection obtains the clotting time.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111707837A (en) * | 2020-05-29 | 2020-09-25 | 上海太阳生物技术有限公司 | Lupus anticoagulant confirmation kit (coagulation method) |
| CN112433057A (en) * | 2020-11-10 | 2021-03-02 | 北京美创新跃医疗器械有限公司 | Screening reagent for lupus anticoagulant and preparation method thereof |
| CN112557665A (en) * | 2020-11-10 | 2021-03-26 | 北京美创新跃医疗器械有限公司 | Confirmation reagent for lupus anticoagulant and preparation method thereof |
| CN114778858A (en) * | 2022-06-23 | 2022-07-22 | 深圳市帝迈生物技术有限公司 | Lupus anticoagulant detection kit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107589251A (en) * | 2017-05-19 | 2018-01-16 | 上海原科实业发展有限公司 | Lupus anticoagulant analyte detection kit and lupus anticoagulant presence or absence determination methods |
-
2018
- 2018-12-07 CN CN201811493765.8A patent/CN109521204A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107589251A (en) * | 2017-05-19 | 2018-01-16 | 上海原科实业发展有限公司 | Lupus anticoagulant analyte detection kit and lupus anticoagulant presence or absence determination methods |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111707837A (en) * | 2020-05-29 | 2020-09-25 | 上海太阳生物技术有限公司 | Lupus anticoagulant confirmation kit (coagulation method) |
| CN112433057A (en) * | 2020-11-10 | 2021-03-02 | 北京美创新跃医疗器械有限公司 | Screening reagent for lupus anticoagulant and preparation method thereof |
| CN112557665A (en) * | 2020-11-10 | 2021-03-26 | 北京美创新跃医疗器械有限公司 | Confirmation reagent for lupus anticoagulant and preparation method thereof |
| CN114778858A (en) * | 2022-06-23 | 2022-07-22 | 深圳市帝迈生物技术有限公司 | Lupus anticoagulant detection kit |
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Application publication date: 20190326 |