CN112433057B - Screening reagent for lupus anticoagulant and preparation method thereof - Google Patents
Screening reagent for lupus anticoagulant and preparation method thereof Download PDFInfo
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- CN112433057B CN112433057B CN202011248230.1A CN202011248230A CN112433057B CN 112433057 B CN112433057 B CN 112433057B CN 202011248230 A CN202011248230 A CN 202011248230A CN 112433057 B CN112433057 B CN 112433057B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of biology, in particular to a screening reagent of lupus anticoagulant and a preparation method thereof. The screening reagent comprises tris (hydroxymethyl) aminomethane, a coagulation factor X activator, rabbit cephalin, gelatin, glycine, trehalose, thimerosal sodium and calcium chloride. The lupus anticoagulant screening kit has good stability and strong anti-interference capability by adjusting the concentration and the components of the stabilizing agent, the reagent can be stabilized for 10 days after being dissolved again at the temperature of 2-8 ℃, and the freeze-dried powder can be stabilized for 2 years after being placed at the temperature of 2-8 ℃. The invention has better stability than the common commercial products, makes full use of the reagent, reduces the reagent waste and saves the cost of the hospital.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a screening reagent of lupus anticoagulant and a preparation method thereof.
Background
Lupus Anticoagulant (LA) is a pathologically circulating anticoagulant, either IgG, igM or a mixture of both. LA blocks the action of activated factor v with prothrombin by recognizing phospholipid binding to prothrombin, inhibits fibrin formation, and interferes with APTT, PT, dRVVT clotting assay in vitro, resulting in prolonged clotting time.
In vivo, LA activates platelets and/or induces pre-thrombotic states by β2-GPI binding, inducing adhesion molecules, tissue factor expression and complement activation, promoting thrombosis. LA acts on vascular endothelial cell membrane phospholipids, interfering with the release of plasminogen activator by endothelial cells to inhibit fibrinolysis; LA acts on platelet membrane phospholipids to interfere with arachidonic acid metabolism and promote platelet activation; LA inhibits endogenous anticoagulant substances associated with phospholipids and enhances blood clotting. About 1/3 of the patients with positive LA tests develop thrombi, and the most common are deep vein thrombosis, pulmonary embolism, and large vessel thrombosis, thrombocytopenia, habitual abortion, stillbirth, and fetal development retardation which are unexplained in obstetrics. Therefore, it is important to provide a reagent for detecting LA.
Detection principle: a screening reagent (LAS) is added into tested blood plasma, a blood coagulation factor X activator directly activates the blood coagulation factor X to become activated blood coagulation factor X, blood is coagulated under the participation of calcium and phospholipid, clotting time is measured, if Lupus Anticoagulant (LA) exists in the blood plasma, the blood plasma clotting time is measured to be beyond a normal reference range, and if the Lupus Anticoagulant (LA) does not exist in the blood plasma, the blood plasma clotting time is measured to be within the normal reference range.
The existing lupus anticoagulant screening reagent has the problem of poor stability, and hemoglobin, bilirubin, triglyceride and heparin in a sample interfere with the accuracy of a detection result.
Disclosure of Invention
In view of the above, the invention provides a screening reagent for lupus anticoagulant and a preparation method thereof. The lupus anticoagulant screening detection reagent (LAS) has the advantages of high stability, strong anti-interference capability on other interfering substances and high sensitivity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a screening reagent of lupus anticoagulant, which comprises a coagulation factor X activator, rabbit cephalin, buffer solution, gelatin, glycine, trehalose, merosal sodium and calcium chloride;
the screening reagent comprises the following components in percentage by weight:
in the invention, the cephalin in the detection reagent is selected from rabbit cephalin, compared with lecithin, pig cephalin, bovine cephalin, soybean phospholipids, peanut phospholipids, synthetic phospholipids and the like, the rabbit cephalin is matched with other reagents in the screening reagent to ensure that the stability of the screening reagent is stronger, so that the reagent has the characteristics of good stability, long shelf life and the like;
the buffer solution in the detection reagent is Tris-HCl buffer solution, compared with phosphate buffer solution, sodium carbonate-sodium bicarbonate buffer solution, citric acid-sodium citrate buffer solution and HEPES buffer solution, the Tris-HCl buffer solution with pH of 7.4 is matched with other reagents in the screening reagent, so that the stability of the screening reagent is stronger, and the reagent has the characteristics of good stability, long shelf life and the like;
in the prior art, the freeze-drying protective agent and the stabilizer generally comprise saccharides (sucrose, trehalose, lactose, glucose and the like), polymers (polyethylene glycol, pvc, gelatin, polyethyleneimine and the like), amino acids (glycine, arginine, alanine, phenylalanine, threonine, lazy amino acid and the like), and the combination of the gelatin, the glycine and the trehalose is preferably used as the freeze-drying protective agent and the stabilizer, and the stability of the screening reagent is higher after being matched with other reagents in the screening reagent, so that the reagent has the characteristics of good stability, long shelf life and the like;
the preservative in the detection reagent is preferably sodium thimerosal, and compared with the preservatives such as sodium azide, sodium thimerosal, potassium sorbate, sodium lactate and the like, the sodium thimerosal is used as the preservative of the invention, and the stability of the screening reagent is stronger after being matched with other reagents in the screening reagent, so that the reagent has the characteristics of good stability, long shelf life and the like.
Preferably, the screening reagent comprises the following components in percentage by weight:
preferably, the screening reagent comprises the following components in percentage by weight:
more preferably, the screening agent comprises the following components in amounts:
preferably, the screening reagent further comprises water, and the 1L screening reagent comprises the following components in percentage by weight:
preferably, the 1L screening reagent comprises the following components in percentage by weight:
preferably, the amounts of the components in the 1L screening reagent are:
more preferably, the 1L screening reagent comprises the following components in amounts:
the invention provides a preparation method of the screening reagent, which comprises the following steps:
uniformly mixing tris (hydroxymethyl) aminomethane, a coagulation factor X activator, rabbit cephalin, gelatin, glycine, trehalose, thimerosal sodium and calcium chloride;
or, firstly, dissolving the tris (hydroxymethyl) aminomethane in water, then adding the coagulation factor X activator, the rabbit cephalin, the gelatin, the glycine, the trehalose, the thimerosal sodium and the calcium chloride, uniformly mixing, and adjusting the pH to 7.5-8.5.
Preferably, the pH is adjusted to 8.0.
In some embodiments of the invention, the screening agent is prepared by: 2-5 g of tris (hydroxymethyl) aminomethane is weighed and dissolved in 800mL of deionized water, fully stirred for 15-20 min, then 80-100U of coagulation factor X activator, 1.0-1.5 mg of rabbit cephalin, 20-25 g of gelatin, 35-40 g of glycine, 10-15 g of trehalose, 0.1-0.5 g of sulfur Liu Gongna and 1.2-1.8 g of calcium chloride are added, stirring is carried out for 20-50 min, the volume is fixed to 1 liter, the pH value is regulated to 8.0, and the liquid is lupus anticoagulant screening detection reagent (LAS).
The invention also provides a use method of the detection reagent or the kit, wherein the blood plasma to be detected is mixed with the detection reagent in a ratio of 1:1, and the time required for coagulation is measured, so that the lupus anticoagulant screening reagent (LAS) detection time of the blood plasma to be detected is obtained.
The invention provides a screening reagent of lupus anticoagulant and a preparation method thereof. The screening reagent comprises tris (hydroxymethyl) aminomethane, a coagulation factor X activator, rabbit cephalin, gelatin, glycine, trehalose, thimerosal sodium and calcium chloride. Compared with the prior art, the technology has the following advantages:
the lupus anticoagulant screening kit has good stability and strong anti-interference capability by adjusting the concentration and the components of the stabilizing agent, the reagent can be stabilized for 10 days after being dissolved again at the temperature of 2-8 ℃, and the freeze-dried powder can be stabilized for 2 years after being placed at the temperature of 2-8 ℃. The invention has better stability than the common commercial products, makes full use of the reagent, reduces the reagent waste and saves the cost of the hospital.
Detailed Description
The invention discloses a screening reagent of lupus anticoagulant and a preparation method thereof, and the technical parameters can be properly improved by a person skilled in the art by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The invention provides a reagent (LAS) for screening and detecting lupus anticoagulant, which is prepared from the following raw materials: 2 to 5g of tris (hydroxymethyl) aminomethane, 80 to 100U of coagulation factor X activator, 1.0 to 1.5mg of rabbit cephalin, 20 to 25g of gelatin, 35 to 40g of glycine, 10 to 15g of trehalose, 0.1 to 0.5g of sulfur Liu Gongna, 1.2 to 1.8g of calcium chloride and the balance of deionized water, wherein the final total volume is 1 liter.
The preparation method comprises the following steps: 2-5 g of tris (hydroxymethyl) aminomethane is weighed and dissolved in 800mL of deionized water, fully stirred for 15-20 min, then 80-100U of coagulation factor X activator, 1.0-1.5 mg of rabbit cephalin, 20-25 g of gelatin, 35-40 g of glycine, 10-15 g of trehalose, 0.1-0.5 g of sulfur Liu Gongna and 1.2-1.8 g of calcium chloride are added, stirring is carried out for 20-50 min, the volume is fixed to 1 liter, the pH value is regulated to 8.0, and the liquid is lupus anticoagulant screening detection reagent (LAS).
The manual detection principle of the reagent comprises the following steps: the plasma to be measured was added to the glass tube, after 1min of incubation at 37℃the pre-Wen Ben inventive reagent was added to the plasma, and the time from addition of the reagent to blood clotting was recorded. The reagent is the lupus anticoagulant screening detection reagent (LAS) time of the plasma to be detected.
TABLE 1 methods of use of screening reagents of the invention
The raw materials and the reagents used in the assay reagent and the application thereof and the kit containing the assay reagent can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
2g of tris (hydroxymethyl) aminomethane is weighed and dissolved in 800mL of deionized water, fully stirred for 15-20 min, then 80U of coagulation factor X activator, 1.0mg of rabbit cephalin, 20g of gelatin, 35g of glycine, 15g of trehalose, liu Gongna 0.1.1 g of sulfur and 1.2g of calcium chloride are added, stirring is carried out for 20-50 min, the volume is fixed to 1 liter, the PH is regulated to 8.0, and the solution is lupus anticoagulant screening detection reagent (LAS).
Example 2
3g of tris (hydroxymethyl) aminomethane is weighed and dissolved in 800mL of deionized water, fully stirred for 15-20 min, then 90U of coagulation factor X activator, 1.2mg of rabbit cephalin, 22g of gelatin, 37g of glycine, 10g of trehalose, liu Gongna 0.3.3 g of sulfur and 1.5g of calcium chloride are added, stirring is carried out for 20-50 min, the volume is fixed to 1 liter, the PH is regulated to 8.0, and the solution is lupus anticoagulant screening detection reagent (LAS).
Example 3
5g of tris (hydroxymethyl) aminomethane is weighed and dissolved in 800mL of deionized water, fully stirred for 15-20 min, then 100U of coagulation factor X activator, 1.5mg of rabbit cephalin, 25g of gelatin, 40g of glycine, 14g of trehalose, liu Gongna 0.5.5 g of sulfur and 1.8g of calcium chloride are added, stirring is carried out for 20-50 min, the volume is fixed to 1 liter, the PH is regulated to 8.0, and the solution is lupus anticoagulant screening detection reagent (LAS).
Test example 1A test kit for screening and detecting Lupus Anticoagulant (LAS) of the present invention and a test for comparing the stability of commercial lupus anticoagulant screening and detecting kit (LAS) in open bottle and reconstitution
1. The lupus anticoagulant screening kit (LAS) and the commercial lupus anticoagulant screening kit (LAS) provided in the embodiment 1 of the invention are re-dissolved in the stability under the condition of 2-8 ℃ after being unsealed
The lupus anticoagulant screening kit (LAS) provided in the embodiment 1 of the invention and the commercial lupus anticoagulant screening kit (LAS) are unsealed and redissolved and then stored at 2-8 ℃, and the test is carried out on a Coatron3000 coagulometer with the same normal quality control (35-50 s) every day, and the results are shown in Table 2.
Table 2 comparison of stability of the open-bottle reconstitution with commercial Lupus Anticoagulant (LA) screening reagents
Note that: commercial lupus anticoagulant confirmation reagent consists of Russell viper venom, phospholipid, an anti-heparin agent, calcium, a buffer solution, a stabilizer, sodium azide and a dye.
Table 2 shows that the stability of the lupus anticoagulant screening reagent (LAS) provided by the embodiment 1 of the invention after bottle opening and reconstitution is obviously better than that of the commercial lupus anticoagulant screening reagent (LAS), the lupus anticoagulant screening reagent (LAS) can be stabilized for 10 days after bottle opening and reconstitution, and the commercial lupus anticoagulant screening reagent (LAS) can be placed for about 2 days.
The lupus anticoagulant screening reagent (LAS) prepared in example 2 and example 3 of the present invention was subjected to the above test, and the results were similar to those of the reagent prepared in example 1, with no significant difference.
The stability of the lupus anticoagulant screening reagent (LAS) provided by the invention is obviously improved.
2. Comparison of the accelerated thermal stability at 37 ℃ after the Lupus anticoagulant screening reagent (LAS) and the commercial Lupus anticoagulant screening reagent (LAS) lyophilized powder provided in example 1 of the present invention are reconstituted
The lupus anticoagulant screening reagent (LAS) provided by the embodiment 1 of the invention is stored for continuous measurement at 37 ℃ for 24 hours after being redissolved, the quality control plasma is detected, the result is stable, the commercial lupus anticoagulant screening reagent (LAS) is changed after 8 hours after being redissolved, and the result shows that the lupus anticoagulant screening reagent (LAS) provided by the embodiment 1 of the invention has good stability and long standing time at 37 ℃, and reduces certain cost and expenditure for hospitals. (the results are shown in Table 3).
TABLE 3 accelerated thermal stability contrast test at 37℃after reconstitution of the lupus anticoagulant screening reagent (LAS) and the commercial lupus anticoagulant screening reagent (LAS) of the present invention
The lupus anticoagulant screening reagent (LAS) prepared in example 2 and example 3 of the present invention was subjected to the above test, and the results were similar to those of the reagent prepared in example 2, with no significant difference. The lupus anticoagulant screening reagent (LAS) provided by the invention has the advantages of good stability and long standing time at 37 ℃, and reduces certain cost and expenditure for hospitals.
Test example 2A test for screening and detecting Lupus Anticoagulant (LAS) and a test for comparing interference resistance of a commercial Lupus anticoagulant screening and detecting kit (LAS)
The lupus anticoagulant screening kit (LAS) has high stability and high anti-interference capability by adjusting the concentration and the components of the stabilizing agent, and the result is not affected when the lupus anticoagulant screening kit contains hemoglobin (0-300 mg/dL), bilirubin (0-20 mg/dL), triglyceride (0-800 mg/dL) and heparin (0-300 IU/dL) in a specimen.
TABLE 4 detection results of interference resistance of commercial lupus anticoagulant screening detection kit (LAS)
TABLE 5 anti-interference performance test results of lupus anticoagulant screening kit (LAS) according to example 1 of the present invention
Test example 3 screening of cephalin required for lupus anticoagulant screening reagent (LAS) of the present invention
In the screening reagent of the present invention, the influence of phospholipids of different sources on the stability of the reagent was examined after the selection of other reagent components, and the quality control plasma was examined at 37℃for 20 hours, and the results are shown in Table 6.
TABLE 6 influence of different types of cephalins on stability of Lupus anticoagulant screening reagent (LAC) at 37℃
As can be seen from Table 6, the stability of lupus anticoagulant screening reagents made with rabbit cephalin was far better than those made with phospholipids from other sources.
Test example 4 Effect of stabilizers and lyoprotectants on stability of Lupus anticoagulant screening reagent (LAS)
1. Effect of different classes of polymers on stability of Lupus anticoagulant screening reagents (LAS)
The 1L lupus anticoagulant screening reagent component comprises: under the conditions of 80U of coagulation factor X activator, 1.0mg of rabbit cephalin, 2g of tris (hydroxymethyl) aminomethane, liu Gongna 0.1.1 g of sulfur and 1.2g of calcium chloride, the influence of the addition of polymer substances on the stability of LAS was examined. 3% of polyethylene glycol, pvc, gelatin and polyethyleneimine are respectively added, the mixture is preserved at 37 ℃, and the quality control plasma is detected at fixed time, and the test results are shown in Table 7.
TABLE 7 influence of addition of different classes of polymers at 37℃on the stability of Lupus anticoagulant screening reagents (LAS)
As can be seen from table 7, gelatin has the best stability effect on lupus anticoagulant screening reagents.
2. Effect of different classes of amino acids on stability of Lupus anticoagulant screening reagents (LAS)
The 1L lupus anticoagulant screening reagent component comprises: under the conditions of 80U as a coagulation factor X activator, 1.0mg of rabbit cephalin, 2g of tris (hydroxymethyl) aminomethane, liu Gongna 0.1.1 g of sulfur and 1.2g of calcium chloride, the effect of the addition of amino acids on the stability of LAS was examined. 3% glycine, arginine, phenylalanine, threonine and lysine (hydrochloride) are respectively added, the mixture is preserved at 37 ℃, and the quality control plasma is detected at regular time, and the test results are shown in Table 8.
TABLE 8 influence of addition of different classes of amino acids at 37℃on the stability of Lupus anticoagulant screening reagents (LAS)
As can be seen from table 8, glycine has the best stability effect on lupus anticoagulant screening reagents.
3. Effect of different classes of saccharides on stability of Lupus anticoagulant screening reagents (LAS)
The 1L lupus anticoagulant screening reagent component comprises: under the conditions of 80U as a coagulation factor X activator, 1.0mg of rabbit cephalin, 2g of tris (hydroxymethyl) aminomethane, liu Gongna 0.1.1 g of sulfur and 1.2g of calcium chloride, the effect of adding saccharide on the stability of LAC was examined. 2% of sucrose, trehalose, lactose, glucose and mannitol are respectively added, the mixture is preserved at 37 ℃, and the quality control plasma is detected at regular time, and the test results are shown in Table 9.
TABLE 9 influence of the addition of different classes of sugar substances at 37℃on the stability of Lupus anticoagulant screening reagents (LAS)
As is clear from Table 9, trehalose has an optimal effect on the stability of the lupus anticoagulant screening reagent.
4. Influence of different concentrations of gelatin on stability of lupus anticoagulant screening reagent (LAS)
The 1L lupus anticoagulant screening reagent component comprises: 80U of coagulation factor X activator, 1.0mg of rabbit cephalin, 2g of tris (hydroxymethyl) aminomethane, liu Gongna 0.1.1 g of sulfur and 1.2g of calcium chloride are added, 3% of glycine and 2% of trehalose are added on the basis of the above, and under the condition, 1% -6% of gelatin is added respectively, so that the influence of gelatin concentration on the stability of LAS is studied. LAS was stored at 37℃and the quality control plasma was examined at regular intervals, and the test results are shown in Table 10.
TABLE 10 influence of gelatin content at 37℃on stability of Lupus anticoagulant screening reagent (LAS)
As can be seen from Table 10, the addition of 1 to 5% gelatin to the components of the screening reagent was effective in ensuring that the lupus anticoagulant screening reagent (LAS) was stable at 37℃for 20 hours.
5. Effect of different concentrations of Glycine on stability of Lupus anticoagulant screening Agents (LAS)
The 1L lupus anticoagulant screening reagent component comprises: 80U of coagulation factor X activator, 1.0mg of rabbit cephalin, 2g of tris (hydroxymethyl) aminomethane, liu Gongna 0.1.1 g of sulfur and 1.2g of calcium chloride are added, 2% of trehalose and 1-5% of gelatin (concentration settings: 1%, 2%, 3%, 4% and 5%) are added on the basis of the above, and under the conditions, 1-6% of glycine is added respectively to investigate the effect of glycine concentration on the stability of LAS. LAS was stored at 37℃and the quality control plasma was examined at regular intervals, and the test results are shown in Table 11.
TABLE 11 Effect of Glycine content at 37℃on stability of Lupus anticoagulant screening reagent (LAS)
As can be seen from Table 11, the selective addition of 1-5% glycine to the components of the screening reagent effectively ensured that the lupus anticoagulant screening reagent (LAS) was stable at 37℃for 20 hours.
6. Effect of different concentrations of trehalose on stability of Lupus anticoagulant screening reagent (LAS)
The 1L lupus anticoagulant screening reagent component comprises: 80U of coagulation factor X activator, 1.0mg of rabbit cephalin, 2g of tris (hydroxymethyl) aminomethane, liu Gongna 0.1.1 g of sulfur and 1.2g of calcium chloride are added, and based on the above, 1-5% of glycine (concentration settings: 1%, 2%, 3%, 4% and 5%) and 1-5% of gelatin (concentration settings: 1%, 2%, 3%, 4% and 5%) are added, under the condition, 0.5-4.0% of trehalose are added respectively, and the influence of the trehalose concentration on the stability of LAS is studied. LAS was stored at 37℃and the quality control plasma was examined at regular intervals, and the test results are shown in Table 12.
TABLE 12 Effect of trehalose content on stability of Lupus anticoagulant screening reagent (LAS) at 37℃
As can be seen from Table 12, the addition of trehalose at 0.5% to 3.0% to the components of the above-mentioned confirmation reagent was effective in ensuring that the lupus anticoagulant screening reagent (LAS) was stable at 37℃for 22 hours.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (3)
1. A screening reagent for lupus anticoagulant, which is characterized by comprising a coagulation factor X activator, rabbit cephalin, buffer solution, gelatin, glycine, trehalose, thimerosal sodium and calcium chloride;
the dosage of each component in 1L of the screening reagent is as follows:
2. the method for preparing a screening reagent according to claim 1, comprising the steps of:
uniformly mixing tris (hydroxymethyl) aminomethane, a coagulation factor X activator, rabbit cephalin, gelatin, glycine, trehalose, thimerosal sodium and calcium chloride;
or, firstly, dissolving the tris (hydroxymethyl) aminomethane in water, then adding the coagulation factor X activator, the rabbit cephalin, the gelatin, the glycine, the trehalose, the thimerosal sodium and the calcium chloride, uniformly mixing, and adjusting the pH to 7.5-8.5.
3. The method of claim 2, wherein the pH is adjusted to 8.0.
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| CN102692516B (en) * | 2012-06-08 | 2015-01-28 | 上海太阳生物技术有限公司 | Lupus anticoagulant (LA) screening and determining reagent kit (freezing method) |
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