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Liquid ready-to-use antithrombin III activity determination reagent

A technology for the determination of antithrombin and activity, applied in the field of biological detection, can solve the problems of high requirements for storage and transportation, differences between freeze-dried powder preparation bottles, and affecting test results, etc., achieving low cost and good stability , The effect of the simple preparation process

Inactive Publication Date: 2017-09-12
NINGBO ACCUTECH BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent CN106153612A discloses a detection reagent for antithrombin III based on FXa. The reagent is relatively stable at 4°C, but it can only be stored for 5 days at 25°C. Carelessness will lead to inactivation of the reagents, which will greatly affect the test results
At present, most of the FXa-based antithrombin III detection reagents on the market use lyophilized powder preparations. However, lyophilized powder reagents require a freeze-drying process, which is relatively complicated to produce, and needs to be reconstituted during use. Due to the freeze-drying process, The difference in the freezing effect of each bottle, as well as the difference in the amount of sample added during the reconstitution process, will lead to differences between bottles of lyophilized powder preparations, and large differences between batches

Method used

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  • Liquid ready-to-use antithrombin III activity determination reagent
  • Liquid ready-to-use antithrombin III activity determination reagent
  • Liquid ready-to-use antithrombin III activity determination reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 reagent 1 and reagent 2

[0028] Preparation of protective agent: use 20mmol / L Tris buffer solution with pH 8.1 to prepare protective agents with different components, add preservative to the protective agent, and the preservative is sodium azide with a working concentration of 0.05% .

[0029] Protectant 1: 2% BSA, 10% sucrose, 2% PEG6000, 10% mannitol.

[0030] Protectant 2: 2% BSA, 10% sucrose, 2% PEG6000.

[0031] Protectant 3: 2% BSA, 2% PEG6000, 10% mannitol.

[0032]Protective agent 4: 10% sucrose, 2% PEG6000, 10% mannitol.

[0033] Preparation of reagent 1: Weigh 2.428g of Tris, 1.02g of sodium chloride, add 900ml of pure water, fully dissolve, adjust the pH to 8.1, add FXa (purchased from SIGMA), and mix slowly until the concentration is 0.4U / ml , the final volume was adjusted to 1000ml with purified water, and equal volumes of solutions of the above-mentioned different protective agents were added respectively, so that the f...

Embodiment 2

[0038] Example 2 standard curve making

[0039] Prepare reagent 1 and reagent 2 according to the method of example 1. Protective agent 3 was selected as the protective agent of reagent 1. Take the antithrombin III standard substance with a concentration of 100% and dilute it with 0.9% normal saline to form a standard solution with a concentration of 50% and 0%. Take 4 μl of the standard solution and add 160 μl of reagent 1 to incubate for 2 minutes, then add 40 μl of Reagent 2 uses a kinetic method to measure the change rate of OD405 absorbance, and uses a logarithmic linear equation to make a standard curve using the change rate of OD405 absorbance and the corresponding antithrombin III concentration, as shown in figure 1 shown.

Embodiment 3

[0040] Example 3 Linear range detection

[0041] Prepare samples with antithrombin III activity concentrations of about 20%, 60%, 100% and 140%, and repeat the detection four times with a Sysmex hemagglutination analyzer CA1500, and take the average value, as shown in Table 2, do Linear regression curve, the linear range is 0~140%, the result figure 2 shown.

[0042] Table 2

[0043]

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Abstract

The invention discloses a liquid ready-to-use antithrombin III activity determination reagent which comprises a reagent 1 and a reagent 2; the reagent 1 is prepared from FXa, heparin, a buffer solution and a protective agent; and the reagent 2 is prepared from a chromogenic substrate of the FXa and mannitol. The liquid ready-to-use antithrombin III activity determination reagent provided by the invention has the advantages of high sensitivity, good stability and convenience in use.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a liquid ready-to-use antithrombin III activity assay reagent and a corresponding kit. Background technique [0002] Blood coagulation is a process regulated by many factors. When blood vessels are damaged, the body activates the coagulation process through internal and external pathways, and finally thrombin is activated, and thrombin decomposes fibrinogen into fibrin, resulting in coagulation. Antithrombin III is a regulatory factor in the coagulation process. Antithrombin III can bind to multiple factors involved in the coagulation process, and inhibit the activity of these coagulation factors, such as FXa, FIXa, FXIa, FIIa. However, in the absence of heparin, the combination of antithrombin III with Fxa and FIIa is very slow. In the presence of heparin, heparin and antithrombin III combine to form a complex, which changes the antithrombin III. The ...

Claims

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Application Information

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IPC IPC(8): G01N21/31
CPCG01N21/31
Inventor 赖增祖叶欣杨奎东徐健
Owner NINGBO ACCUTECH BIOSCI LTD
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