阅读说明：本技术 抽提缓冲液、兔脑抽提液、pt检测试剂及pt检测试剂盒 (Extraction buffer solution, rabbit brain extract, PT detection reagent and PT detection kit ) 是由 丁重辉 胡晓娟 于 2019-11-29 设计创作，主要内容包括：本发明涉及医学检测领域,特别涉及抽提缓冲液、兔脑抽提液、PT检测试剂及PT检测试剂盒。本发明提供的抽提缓冲液包括柠檬酸盐或磷酸盐、无水乙酸钠和水。本发明通过外加电解质,增加了磷脂间电荷的斥力,使PT试剂不容易形成沉淀,更加稳定,测试时的重复性更好。(The invention relates to the field of medical detection, in particular to an extraction buffer solution, a rabbit brain extract, a PT detection reagent and a PT detection kit. The extraction buffer provided by the invention comprises citrate or phosphate, anhydrous sodium acetate and water. According to the invention, through adding the electrolyte, the repulsion of charges among phospholipids is increased, so that the PT reagent is not easy to form precipitates, is more stable and has better repeatability in testing.)

1. The extraction buffer solution of the rabbit brain powder is characterized by comprising the following components in percentage by mass:

2. the extraction buffer of claim 1, comprising the following components in mass percent:

3. the extraction buffer according to claim 1 or 2, wherein the citrate is trisodium citrate; the phosphate comprises one or more of sodium phosphate, potassium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate or dipotassium hydrogen phosphate.

4. The extraction buffer according to any of claims 1 to 3, wherein the pH is adjusted with acetic acid.

5. Use of an extraction buffer according to any one of claims 1 to 4 for the preparation of rabbit brain extract.

6. Use of the extraction buffer according to any of claims 1 to 4 for the preparation of a PT detection reagent or a PT detection kit.

7. The rabbit brain extract is characterized by comprising the following steps: dissolving rabbit brain powder in the extraction buffer solution of any one of claims 1 to 4, and incubating at 33-45 ℃ for 0.5-1.5 h;

and the mass-volume ratio of the rabbit brain powder to the extraction buffer solution is (3-5): 100 in g/mL.

8. The rabbit brain extract according to claim 7, wherein the incubation is performed with shaking every 15min, and the incubation further comprises the step of collecting the supernatant after centrifugation at 3000 RPM.

A PT detection reagent comprising the rabbit brain extract according to claim 7 or 8, a lyoprotectant, a heparin antagonist, and calcium ions;

the freeze-drying protective agent comprises one or a mixture of more than two of bovine serum albumin, peptone, glycine, alanine, sucrose and trehalose; the mass percentage of the bovine serum albumin and the peptone is 0.5 to 5 percent, and the mass percentage of the glycine, the alanine, the sucrose and the trehalose is 0.1 to 10 percent;

the heparin antagonist comprises one or two of polybrene and protamine; the concentration of the heparin antagonist is 0.01mg/ml-0.1 mg/ml;

the agent for providing calcium ions comprises one or more of calcium chloride, calcium lactate or calcium carbonate; the concentration of the calcium ions is 10 mM-25 mM.

A PT assay kit comprising the extraction buffer of any one of claims 1 to 4, the rabbit brain extract of claim 7 or 8, or the PT assay reagent of claim 9.

Technical Field

The invention relates to the field of medical detection, in particular to an extraction buffer solution, a rabbit brain extract, a PT detection reagent and a PT detection kit.

Background

Clinically, it is necessary to detect the blood coagulation function of a patient in order to know whether the hemostatic function of the patient is defective or not before an operation, to prepare in advance and to prevent a large bleeding during the operation, and Prothrombin Time (PT) is one of the methods for detecting the blood coagulation function. PT is a main way for detecting the deficiency of exogenous coagulation factors, is used for confirming the defects or the existence of inhibitors of congenital or acquired fibrinogen, prothrombin and coagulation factors V, VII and X, is used for detecting the dosage of oral anticoagulants, and is a first-choice index for monitoring the oral anticoagulants.

The main component of the PT reagent is tissue thromboplastin (i.e. a mixture of tissue factor and phospholipid), some researches suggest that rabbit brain powder is obtained by processing fresh rabbit brain, and the rabbit brain powder is thermally activated in physiological saline at 37 ℃, then centrifuged to obtain supernatant, and freeze-dried to obtain a freeze-dried preparation of the rabbit brain tissue factor, but the patent does not mention how to improve the suspension property of the freeze-dried preparation of the rabbit brain tissue factor. Other scholars have mentioned the preparation of in vitro diagnostic kits for measuring PT by using rabbit brain powder as a raw material, but have not mentioned how to improve the suspension property of the PT reagent, and the suspension property directly influences the repeatability of the reagent.

The PT reagent derived from rabbit brain powder is prepared by infiltrating two coagulation promoting substances, namely tissue factor and phospholipid, contained in the rabbit brain powder in the form of solution, and the two substances form the main raw materials of the PT reagent. In the actual production process, the extract of the rabbit brain powder contains not only two substances of tissue factor and phospholipid, but also other non-essential components such as protein and the like, the existence of the non-essential components causes the solute concentration of the extract of the rabbit brain powder to be overhigh, and on the other hand, the extract of the rabbit brain contains a large amount of phospholipid which is far beyond the optimal amount required for solidifying the PT reagent. Too high a solute concentration and the presence of large amounts of phospholipids make this PT reagent less stable, i.e. prone to precipitate formation.

Therefore, it is important to provide a PT reagent having good stability.

Disclosure of Invention

In view of the above, the present invention provides an extraction buffer, a rabbit brain extract, a PT detection reagent, and a PT detection kit. According to the invention, through adding the electrolyte, the repulsion of charges among phospholipids is increased, so that the PT reagent is not easy to form precipitates, is more stable and has better repeatability in testing.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an extraction buffer solution of rabbit brain powder, which comprises the following components in percentage by mass:

in some embodiments of the present invention, the composition comprises the following components by mass percent:

in some embodiments of the invention, the citrate salt is trisodium citrate; the phosphate comprises one or more of sodium phosphate, potassium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate or dipotassium hydrogen phosphate.

In some embodiments of the invention, the pH is adjusted with acetic acid.

The extracted rabbit brain extract belongs to colloid with negative charge on the surface due to the relationship of phospholipid, the stability of the colloid is conditional, once the stable condition is destroyed, the particles in the sol can aggregate and grow up, and finally aggregate and precipitate from the medium, and the factors influencing the aggregation of the colloid comprise heating, electrolyte and the like. Because the leaching solution of the rabbit brain has negative charges, only low-concentration high-valence anions can be added to repel each other, the stability of colloidal particles is kept, and water or normal saline or high-valence cations cannot be used. Therefore, trisodium citrate is added, and since trisodium citrate is very basic, the pH is lowered by adding anhydrous sodium acetate, which is acidic, while the pH is adjusted by acetic acid. The higher anionic compound that may replace trisodium citrate may be a phosphoric acid compound such as sodium phosphate, potassium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, etc.

On the basis of the research, the invention also provides the application of the extraction buffer solution in preparing rabbit brain extraction liquid.

The invention also provides application of the extraction buffer solution in preparation of a PT detection reagent or a PT detection kit.

On the basis of the research, the invention also provides rabbit brain extract, and the preparation method comprises the following steps: dissolving rabbit brain powder in the extraction buffer solution of any one of claims 1 to 4, and incubating at 33-45 ℃ for 0.5-1.5 h;

and the mass-volume ratio of the rabbit brain powder to the extraction buffer solution is (3-5): 100 in g/mL.

In some embodiments of the invention, the incubation is performed by shaking every 15min, and the incubation is followed by a step of collecting the supernatant after centrifugation at 3000 RPM.

The invention also provides a PT detection reagent, which comprises the rabbit brain extract, a freeze-drying protective agent, a heparin antagonist and calcium ions;

the freeze-drying protective agent comprises one or a mixture of more than two of bovine serum albumin, peptone, glycine, alanine, sucrose and trehalose; the mass percentage of the bovine serum albumin and the peptone is 0.5 to 5 percent, and the mass percentage of the glycine, the alanine, the sucrose and the trehalose is 0.1 to 10 percent;

the polybrene added into the reagent can antagonize heparin, so that the PT reagent is not influenced by the heparin during testing, and can only monitor the oral warfarin. The heparin antagonist comprises one or two of polybrene and protamine; the concentration of the heparin antagonist is 0.01mg/ml-0.1 mg/ml;

the agent for providing calcium ions comprises one or more of calcium chloride, calcium lactate or calcium carbonate; the concentration of the calcium ions is 10 mM-25 mM.

The invention also provides a PT detection kit, which comprises the extraction buffer solution, the rabbit brain extract or the PT detection reagent.

The extraction buffer provided by the invention comprises citrate or phosphate, anhydrous sodium acetate and water. According to the invention, through adding the electrolyte, the repulsion of charges among phospholipids is increased, so that the PT reagent is not easy to form precipitates, is more stable and has better repeatability in testing.

Detailed Description

The invention discloses an extraction buffer solution, rabbit brain extract, a PT detection reagent and a PT detection kit. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention provides the following scheme:

1. preparing an extraction buffer solution

The concentration of trisodium citrate and anhydrous sodium acetate may be in a range, 0.1% to 1%.

2. Obtaining extract of rabbit brain

Dissolving 3-5% rabbit brain powder (purchased commercially, mass-to-volume ratio, unit is g/ml) by using the prepared extraction buffer solution, wherein the incubation temperature can be in a certain range and can be 33-45 ℃, the higher the temperature is, the longer the second value of normal quality control is measured, the lower the temperature is, the shorter the second value of normal quality control is measured, the incubation can be adjusted, the range can be 30 min-1.5 h, the longer the time is, the longer the second value of normal quality control is measured, the shorter the time is, and the shorter the second value of normal quality control is measured.

3. Addition of lyoprotectants and heparin antagonists

Adding lyophilized protectant, heparin antagonist and calcium ion into rabbit brain extractive solution. The protective agent can be macromolecular proteins such as bovine serum albumin, peptone and the like, and the concentration is in the range of 0.5-5%; or may be small molecular protein such as glycine, alanine, etc., or small molecular saccharide, with concentration range of 0.1% -10%. The heparin antagonist can be polybrene and protamine, and the concentration is in the range of 0.01mg/ml-0.1 mg/ml. Calcium ion is added, such as calcium chloride, calcium lactate, and calcium carbonate, and the concentration of calcium ion is 10-25 mM.

According to the invention, through adding the electrolyte, the repulsion of charges among phospholipids is increased, so that the PT reagent is not easy to form precipitates, is more stable and has better repeatability in testing.

The raw materials and reagents used in the extraction buffer solution, the rabbit brain extract, the PT detection reagent and the PT detection kit provided by the invention can be purchased from the market.

The invention is further illustrated by the following examples: