CN116859032A - A kind of base liquid of lupus anticoagulant reagent and preparation method of screening confirmation reagent - Google Patents

Abstract

The invention relates to the field of biotechnology, and discloses a base solution of a lupus anticoagulant reagent and a preparation method of a screening confirmation reagent, wherein 20% glycine and 50mM-CaCl are respectively packaged in the base solution and the screening confirmation reagent ₂ 5% mannitol, 1.5% BSA, 0.5% proclin-300, 1M Tris, screening and validation reagents additionally contained 2.5U/ml RVV-Factor X Activator clotting factor X activator, 25mg/ml synthetic phospholipid mixture. The preparation method of the base solution of the lupus anticoagulant reagent and the screening confirmation reagent optimizes the pH value to be between 6.11 and 6.49 by adjusting the components of the base solution, so that the base solution used by the lupus anticoagulant screening confirmation reagent can be stored in a liquid form for a longer period of time more stably, and the reagent has good stability and strong anti-interference capability at the temperature of 2-8 ℃. The prepared screening confirmation reagent can be placed at the temperature of 2-8 ℃ for 2 years in a freeze-dried state, and the stability of the reagent is superior to that of common products sold on the market, so that the reagent is widely applied.

Description

A kind of base liquid of lupus anticoagulant reagent and preparation method of screening confirmation reagent

Technical field

The invention relates to the technical field of biotechnology, and specifically relates to a base solution of a lupus anticoagulant reagent and a preparation method of a screening confirmation reagent.

Background technique

Lupus anticoagulant (LA) is an antiphospholipid antibody found in patients with lupus. The presence of LA increases the clotting ability of blood. Therefore, if you have this antibody, you have a greater risk of blood clotting. Lupus anticoagulant (LA) is a pathological circulating anticoagulant, which is IgG, IgM or a mixture of both. LA binds to prothrombin by recognizing phospholipids, blocking the interaction between activated coagulation factor V and prothrombin, and inhibiting the formation of fibrin. About 30% of lupus patients have anti-phospholipid antibodies. According to research from the Johns Hopkins Lupus Center, antiphospholipid antibodies can directly destroy ① phospholipids in cell membrane components; ② certain blood proteins that bind to phospholipids; and ③ the complex formed when proteins and phospholipids combine. Antiphospholipid antibodies interfere with the normal function of blood vessels and can cause vasoconstriction or blood clots.

In vivo, LA can activate platelets and/or bind to β2-GPI, inducing the expression of adhesion molecules, tissue factor and complement activation to produce a prothrombotic state and promote thrombosis. LA interacts with vascular endothelial cell membrane phospholipids, interfering with the release of plasminogen activator by endothelial cells and inhibiting fibrinolysis; LA interacts with platelet membrane phospholipids, interfering with arachidonic acid metabolism and promoting platelet activation; LA can inhibit endogenous phospholipid-related Anticoagulant substance that enhances blood coagulation. Thrombosis occurs in patients with a positive LA test. The most common ones are deep vein thrombosis, pulmonary embolism and large vessel thrombosis, thrombocytopenia, and obstetric manifestations such as unexplained habitual abortion, stillbirth, and fetal development retardation.

It will interfere with APTT, PT, and dRVVT coagulation tests in vitro, resulting in prolonged coagulation time. These complications can lead to stroke, heart attack, and miscarriage. Therefore, it is of great practical significance to provide a detection reagent for LA.

Contents of the invention

(1) Technical problems solved

In view of the shortcomings of the existing technology, the present invention provides a base solution of a lupus anticoagulant reagent and a preparation method of a screening confirmation reagent, which have the advantages of simple ingredients, easy acquisition, strong stability, and easy storage.

(2) Technical solutions

In order to achieve the above object, the present invention provides the following technical solution: a base liquid of lupus anticoagulant reagent, the base liquid is composed of glycine, calcium chloride, mannitol, BSA, proclin-300, Tris (trihydroxymethylaminomethane) Composed of:

serial number Material name Dosage 1 Glycine 200g 2 CaCl ₂ 5.55g 3 Mannitol 50g 4 BSA 15g 5 proclin-300 5ml 6 1M Tris 0.8ml

Preferably, the base solution of the lupus anticoagulant reagent is made from the following raw materials: glycine 20%, calcium chloride (25mM-CaCl ₂ ) 0.56%, mannitol 5%, BSA 1.5%, proclin-300 0.5% , Tris 0.1%, the balance is water.

Preferably, the pH value of the base solution of the lupus anticoagulant reagent is between 6.11 and 6.49.

The present invention provides a method for preparing a lupus anticoagulant reagent screening confirmation reagent. The screening confirmation reagent includes a screening reagent and a confirmation reagent;

Screening reagents include glycine 20%, calcium chloride (25mM- _CaCl2 ) 0.56%, mannitol 5%, BSA 1.5%, proclin-300 0.5%, Tris 0.01%, RVV Factor Snake venom) 0.9U/L, synthetic phospholipid 25mg/L, the balance is water, the following table is the specific dosage of each component in 1L screening reagent

serial number Material name Dosage 1 Lupus anticoagulant reagent base solution 1000ml 2 2.5U/ml RVV Factor X Activator 360ul 3 25mg/ml synthetic phospholipid mixture 1ml

Confirmation reagents include glycine 20%, calcium chloride (25mM- _CaCl2 ) 0.56%, mannitol 5%, BSA 1.5%, proclin-300 0.5%, Tris 0.01%, RVV Factor Snake venom) 0.9U/L, synthetic phospholipid 25mg/L, the balance is water, the following table is the specific dosage of 1L confirmation reagent

serial number Material name Dosage 1 Lupus anticoagulant reagent base solution 1000ml 2 2.5U/ml RVV Factor X Activator 360ul 3 25mg/ml synthetic phospholipid mixture 10ml

Preferably, the screening reagent preparation method includes the following steps:

Step 1: Measure 500ml of lupus anticoagulant base solution into a beaker, add 180ul of 2.5U/ml RVV Factor XActivator and 0.5ml of 25mg/ml synthetic phospholipid mixture, stir evenly with a magnetic stirrer, and let stand overnight at 2-8°C. use.

Step 2: Mix the overnight screening reagent and aliquot it at room temperature, 2 ml per bottle.

Step three: freeze-dry the packed screening reagents in a freeze-drying machine.

Step 4: Store the freeze-dried screening reagent at 2-8°C.

Preferably, the confirmation reagent preparation method includes the following steps:

Step 1: Measure 500ml of lupus anticoagulant base solution into a beaker, add 180ul of 2.5U/ml RVV Factor XActivator and 5ml of 25mg/ml synthetic phospholipid mixture, stir evenly with a magnetic stirrer, and set aside.

Step 2: After confirming that the reagents are evenly mixed, aliquot 2 ml per bottle at room temperature.

Step 3: freeze-dry the packed confirmation reagent in a freeze-drying machine.

Step 4: Store the freeze-dried confirmation reagent at 2-8°C.

Preferably, the source of the synthetic phospholipid mixture used in the lupus anticoagulant screening confirmation reagent has no biological activity and is synthesized by pure chemical means. The ratio of the synthetic phospholipid mixture is PE:PS:PC=5:3:2.

Preferably, the usage amount of the synthetic phospholipid in the screening reagent and the confirmation reagent is 1:10.

Preferably, the ratio of sample to screening confirmation reagent is 1:1.

Compared with the prior art, the present invention provides a base solution for lupus anticoagulant reagent and a preparation method for screening confirmation reagent, which have the following beneficial effects:

1. The lupus anticoagulant reagent base liquid used in the screening reagent or confirmation reagent of the present invention has simple composition, is easy to obtain, has strong stability, and is easy to store, and can be configured into a corresponding screening reagent or confirmation reagent lyophilized product. Stable at 2-8℃ for 2 years. The base fluid used in screening reagents or confirmation reagents is consistent, making the production process simpler.

2. The phospholipids used in the present invention are pure chemically synthesized phospholipids, which have no source of biological activity and are more stable in composition, effectively reducing the problems of batch-to-batch variation and bottle-to-bottle variation, and effectively ensuring the stability of the reagent.

3. The ratio of reagents and samples used in the detection of the present invention is 1:1, which better alleviates the non-uniform pH between the reagents and samples, and the pH value during the reaction approaches the neutral pH of 7.0.

Detailed ways

The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

A base solution for lupus anticoagulant reagent. The base solution ingredients are glycine 20%, calcium chloride (25mM-CaCl ₂ ) 0.56%, mannitol 5%, BSA 1.5%, proclin-300 0.5%, Tris 0.01% , the remainder is water.

Prepare 1L lupus anticoagulant base solution:

Weigh 200g of glycine, 5.55g of calcium chloride, 50g of mannitol, and 15g of BSA, add them to the beaker, then add 1L of purified water, pipette to absorb 5ml of proclin-300, 0.8ml of 1M Tris, stir well, pH 6.45, spare.

Prepare 100ml snake venom dilution buffer:

S1, 0.1M Tris formulation:

Use an electronic balance to weigh 1.2114g Tris (trihydroxymethylaminomethane) (molecular weight 121.14), and adjust the volume to 100mL with ultrapure water.

S2, 0.1M HCl preparation:

Use a 1ml pipette to measure 0.867ml of concentrated hydrochloric acid (mass fraction 36%) and add ultrapure water to adjust the volume to 100ml.

S3, 0.05M Tris-HCl buffer preparation:

Take 50ml of 0.1mol/L Tris (trishydroxymethylaminomethane) solution and mix it with 44.7ml of 0.1mol/L hydrochloric acid, then add ultrapure water to adjust the volume to 100ml to prepare a solution with pH 7.20.

S4, 2M NaOH preparation:

Weigh 8g NaOH (molecular weight 40), adjust the volume to 100mL with ultrapure water, mix well and set aside.

S5, 0.02M Tris-HCl-0.15M NaCl (pH 7.2) buffer preparation:

Measure 40ml of 0.05M Tris-HCl buffer, add 60ml of purified water and mix well to obtain 100ml of 0.02MTris-HCl buffer. Use 2M NaOH to adjust the pH to a final pH of 7.2 (note that the volume of NaOH used for adjustment cannot exceed the total 3% of the volume), then add 0.8775g NaCl and mix well before use.

Prepare 2.5U RVV-X snake venom solution:

Take 2 ml of 0.02M Tris-HCl-0.15M NaCl (pH 7.2) buffer and add it to the vial of RVV Factor XActivator 5U. Invert and mix well before use.

Prepare 25mg/ml synthetic phospholipid mixture PE:PS:PC=5:3:2 suspension:

Pipette 1 ml of ultrapure water into a vial of synthetic phospholipid mixture PE:PS:PC=5:3:2, shake and mix in a water bath and set aside. If the usage amount is large, multiple bottles must be mixed before use.

Prepare screening reagents:

Step 1: Measure 500ml of lupus anticoagulant base solution into a beaker, add 180ul of 2.5U/ml RVV Factor XActivator and 0.5ml of 25mg/ml synthetic phospholipid mixture, stir evenly with a magnetic stirrer, and let stand overnight at 2-8°C. use.

Step 2: Mix the overnight screening reagent and aliquot it at room temperature, 2 ml per bottle.

Step three: freeze-dry the packed screening reagents in a freeze-drying machine.

Step 4: Store the freeze-dried screening reagent at 2-8°C.

Prepare confirmation reagents:

Step 1: Measure 500ml of lupus anticoagulant base solution into a beaker, add 180ul of 2.5U/ml RVV Factor XActivator and 5ml of 25mg/ml synthetic phospholipid mixture, stir evenly with a magnetic stirrer, and set aside.

Step 2: After confirming that the reagents are evenly mixed, aliquot 2 ml per bottle at room temperature.

Step 3: freeze-dry the packed confirmation reagent in a freeze-drying machine.

Step 4: Store the freeze-dried confirmation reagent at 2-8°C.

After the preparation of the screening reagents in the confirmation kit is completed, STAGO's quality control products LA1+LA2 and the fully automatic coagulation instrument are used to test the samples to be tested. The process is as follows: Place the screening reagents and confirmation reagents into the corresponding reagent positions, and place the plasma to be tested. On the sample rack, take 75ul of the plasma to be tested and pre-warm it at 37°C for 3 minutes, then add 75ul of screening reagent for detection and measure the coagulation time. When the coagulation time is within the reference range given by LA1, it means that the plasma to be tested does not contain lupus. Anticoagulant; when the coagulation time is within the reference range given by LA2, it indicates that the plasma to be tested contains lupus anticoagulant, and a confirmation reagent is needed for confirmation. Take 75ul of the plasma to be tested and prewarm it at 37°C for 3 minutes at the pre-warming position, and then add it to confirm Use 75ul of reagent for testing, measure the coagulation time, and observe whether the coagulation time is significantly shortened.

Calculate the ratio of the screening reagent to the confirmation reagent, compare it with the normal plasma coagulation time ratio reference range, determine the presence or absence of lupus anticoagulant, and determine the normal plasma coagulation time reference range: Collect 30 healthy individuals aged 18-55 years old Plasma, according to the above detection method, use the screening reagent of the present invention to measure the plasma coagulation time of each sample. The measured reference range of normal plasma coagulation time is: 27-41s.

Determination of the reference range of the normal plasma coagulation time ratio: Use the confirmation reagent of the present invention to measure the coagulation time of the above 30 healthy normal plasmas, and compare the normal plasma screening reagent coagulation time with the confirmation reagent coagulation time to obtain the normal plasma coagulation time ratio reference The range is 0.9-1.2.

Store the screening reagents and confirmation reagents at 4°C and 37°C. Take them out when used and reconstitute 2 ml of purified water. Measure the coagulation time of the same LA normal plasma and LA abnormal plasma at different times. The results are shown in the table below:

In summary, the screening reagent and confirmation reagent of the present invention were measured at 4°C and 37°C according to the stage time for 30 days respectively, and the results did not change much, indicating that the reagent of the present invention has good stability and is convenient for measurement.

Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art will understand that various changes, modifications, and substitutions can be made to these embodiments without departing from the principles and spirit of the invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (10)

1. A base solution of a lupus anticoagulant reagent, which is characterized in that: the base solution consists of glycine, calcium chloride, mannitol, BSA, proclin-300 and Tris (Tris (hydroxymethyl aminomethane).

2. The base fluid of a lupus anticoagulant reagent of claim 1 wherein: the base solution of the lupus anticoagulant reagent is prepared from the following raw materials: glycine 20%, calcium chloride (50 mM-CaCl) ₂ ) 0.56%, mannitol 5%, BSA 1.5%, proclin-300.5%, tris0.01% and the balance water.

3. The base fluid of a lupus anticoagulant reagent of claim 1 wherein: the pH value of the base solution of the lupus anticoagulant reagent is between 6.11 and 6.49.

4. The base fluid of a lupus anticoagulant reagent of claim 1 wherein: the basic solution of the lupus anticoagulant reagent 1L comprises the following components in parts by weight: glycine 200g, calcium chloride 5.55g, mannitol 50g,BSA 15g,proclin-300 5ml,1M Tris 0.8ml.

5. A preparation method of a reagent for screening and confirming lupus anticoagulant comprises a reagent base solution of lupus anticoagulant, and is characterized in that: the screening validation reagent includes a screening reagent and a validation reagent;

screening reagents included glycine 20%, calcium chloride (50 m M-CaCl) ₂ ) 0.56 percent of mannitol, 5 percent of BSA, 1.5 percent of proclin-300.5 percent, 0.01 percent of Tris, 0.9U/L of RVV Factor X Activator coagulation factor X activator (viper venom), 25mg/L of synthetic phospholipid and the balance of water;

the validation reagent comprises glycine 20% and calcium chloride(50m M-CaCl ₂ ) 0.56%, 5% mannitol, 1.5% BSA, 0.5% proclin-300, 0.01% Tris, 0.9U/L RVV Factor X Activator coagulation factor X activator (viper venom), 250mg/L synthetic phospholipid and the balance water.

6. The method for preparing a reagent for screening and confirming lupus anticoagulant as set forth in claim 5, wherein: the preparation method of the screening reagent comprises the following steps:

step one, weighing 500ml of lupus anticoagulant base solution, adding 0.5ml of a mixture of 2.5U/ml RVV Factor X Activator 180ul and 25mg/ml synthetic phospholipid into a beaker, uniformly stirring by a magnetic stirrer, and standing at 2-8 ℃ for later use.

Step two, after the overnight screening reagent is uniformly mixed, split charging is carried out at room temperature, and each bottle is 2ml.

And step three, freeze-drying treatment is carried out on the packaged screening reagent freeze dryer.

And step four, storing the lyophilized screening reagent at 2-8 ℃.

7. The method for preparing a reagent for screening and confirming lupus anticoagulant as set forth in claim 5, wherein: the preparation method of the confirmation reagent comprises the following steps:

step one, 500ml of lupus anticoagulant base solution is measured and placed in a beaker, and 5ml of a mixture of 2.5U/ml RVV Factor X Activator ul and 25mg/ml of synthetic phospholipid is added, and the mixture is stirred uniformly by a magnetic stirrer for later use.

Step two, after confirming that the reagent is uniformly mixed, split charging is carried out at room temperature, and each bottle is 2ml.

And thirdly, freeze-drying the packaged confirmation reagent by a freeze dryer.

And step four, preserving the lyophilized confirmation reagent at 2-8 ℃.

8. The method for preparing a reagent for screening and confirming lupus anticoagulant as set forth in claim 6 or 7, wherein: the lupus anticoagulant screening confirms that the source of the synthetic phospholipid mixture used in the reagent is not bioactive, and the synthetic phospholipid mixture is synthesized by adopting a pure method, wherein the proportion of the synthetic phospholipid mixture is PE: PS: PC=5:3:2.

9. The method for preparing a reagent for screening and confirming lupus anticoagulant as set forth in claim 8, wherein: the amount of the synthetic phospholipid used in the screening reagent and the confirmation reagent is 1:10.

10. The method for preparing a reagent for screening and confirming lupus anticoagulant as set forth in claim 5, wherein: the ratio of sample to screen confirmation reagent was 1:1.