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Kit (Developing substrate method) for testing antithrombase III (AT-III)

A technology of antithrombin and kits, applied in biochemical equipment and methods, microbial measurement/testing, fermentation, etc., can solve the problems that are not suitable for automatic analyzers, analysis requires a large amount of dilution, FXa is not stable, etc. , to achieve the effect of wide detection linear range, wide application and high sensitivity

Active Publication Date: 2015-01-28
SHANGHAI SUNBIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Demers et al. (Thrombosis and Hemostasia, 69 (3), pp.231-235 (1993)) reported that using FXa instead of thrombin will not be inhibited by HCII, but the analysis with FXa is more expensive and FXa is not stable. Analysis requires a lot of dilution, not suitable for automatic analyzers

Method used

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  • Kit (Developing substrate method) for testing antithrombase III (AT-III)
  • Kit (Developing substrate method) for testing antithrombase III (AT-III)
  • Kit (Developing substrate method) for testing antithrombase III (AT-III)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 Preparation of Heparin Derivatives

[0047] Step 1: Dissolve heparin (purchased from sigma company, product number: H3393, activity 190 USP / mg) in 50 mM phosphate buffer solution with pH value 7.0 to prepare a heparin stock solution with a concentration of 2000 U / ml.

[0048] Step 2: Heparanase II (purchased from Beijing Adhawk International Technology Co., Ltd., product number HS6512, 9.8IU / mg) was redissolved in distilled water to a concentration of 20IU / ml. Add the heparanase II solution to the heparin mother liquor obtained in step 1, and the concentration ratio of heparanase II and heparin in the mixture is 1IU:8000U.

[0049] After the mixture was incubated under airtight conditions at 30°C for 30 hours, the absorbance was measured at 232nm. Using phosphate buffer as a blank control, it increased by 15% compared to the original value, indicating that the enzyme digestion reaction had occurred and the unsaturated disaccharides had been removed from the...

Embodiment 2

[0051] Composition and preparation method of embodiment 2 detection kit

[0052] R1 reagent is mainly prepared from the following raw materials: bovine thrombin concentration is 60IU / ml, heparin derivative (made in Example 1) is 9.0U / ml, Tris is 10mM, Tween-80 is 0.2mg / ml, gelatin 0.05mg / ml, BSA 1.2mg / ml, polyethylene glycol-8000 5mg / ml and sodium azide 0.5mg / ml, lyophilized in 1ml / bottle.

[0053] R1 Reagent Reconstitution Reagent is prepared from the following raw materials: Tris concentration is 40mM, KCl is 0.2M, EDTA-K2 is 3.75mM, NaN3 is 0.5mg / ml, and the pH value is adjusted to 8.40 with 1mol / L HCl at 25°C.

[0054] R2 Reagent is formulated from: Chromogenic Substrate

[0055] H-D-Phe-Pip-Arg-pNA·2HCl is 3.6 μmol / ml, mannitol is 30 mg / ml, sodium azide is 3 mg / ml, and lyophilized in 1 ml / bottle.

[0056] The AT-III calibrator is to mix the collected healthy normal human plasma, add glycine and sodium azide to dissolve, and adjust the pH value to 7.5, the concentration ...

Embodiment 3

[0057] The assay method of embodiment 3 detection kits

[0058] (1) Reconstitute the R1 reagent, R2 reagent and AT-III standard obtained in Example 2:

[0059] Reconstitute each bottle of R1 reagent with 3ml R1 reconstitution reagent, each bottle of R2 reagent with 3ml distilled water, and each bottle of AT-Ⅲ calibrator with 1ml distilled water.

[0060] (2) Taking the operation of CA530 hemagglutination analyzer in East Asia, Japan as an example, set the analysis program according to the instrument instructions: the measurement wavelength is 405nm;

[0061] Take 5 μl of plasma sample, add 120 μl of normal saline to dilute, the dilution ratio is 1:25, incubate at 37°C for 30 seconds, take 50 μl of the diluted sample, add 60 μl of R1 reagent and incubate at 37°C for 90 seconds, then add 60 μl of R2 reagent and incubate at 37°C After incubation, measure the difference in absorbance (△OD) between the 10th second and the 40th second. The instrument automatically dilutes the cali...

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Abstract

The invention discloses a kit for detecting antithrombase III (AT-III) in blood plasma by a developing substrate method. The kit comprises a heparin derivative, thrombin and a developing substrate reagent, wherein the heparin derivative is obtained by degrading heparin through heparinase II and cracking one or more unsaturated disaccharides from the heparin. The detection kit disclosed by the invention can avoid interference of a heparin cofactor II (HC-II) in a blood plasma sample, has the advantages of strong anti-interference capability, high flexibility, wide linear range, simplicity and rapidness in operation, strong instrument compatibility and the like, and has a wide application prospect.

Description

technical field [0001] The invention relates to the field of medical in vitro diagnosis, in particular to a kit for detecting antithrombin III by a chromogenic substrate method. Background technique [0002] Antithrombin III (antithrombin III, AT-III) is a heparin-dependent serine protease inhibitor and an important anticoagulant factor, responsible for 60% to 70% of antithrombin in plasma It plays an important role in maintaining the balance of blood physiological coagulation and anticoagulation. AT-III is synthesized by the liver, vascular endothelial cells and megakaryocytes, with a molecular weight of about 60,000 Da, belonging to α2-globulin, and its gene is located on chromosome 1 (1p23). [0003] In the process of thrombus formation, AT-III is a very important regulator. Under the catalysis of heparin, AT-III is catalyzed by thrombin or coagulation factors IXa, Xa, XIa, XIIa, plasmin and other serine proteases in a ratio of 1:1. Complexes are formed to inactivate th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/56C12P19/12
Inventor 谢永华
Owner SHANGHAI SUNBIO TECH
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