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

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

Active Publication Date: 2012-09-26
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] Example 1 Preparation of heparin derivatives

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

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

[0049] After the mixed solution was incubated for 30 hours under airtight conditions at 30°C, the absorbance was measured at 232nm. Phosphate buffer was used as a blank control, which increased by 15% from the original value, indicating that the enzyme digestion reaction has occurred and the unsaturated disaccharide has been removed from heparin. Cleavage to form he...

Embodiment 2

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

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

[0053] The R1 reagent reconstitution reagent is prepared from the following 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 at 25°C with 1mol / L HCl.

[0054] R2 reagent is prepared from the following reagents: chromogenic substrate

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

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

Embodiment 3

[0057] Example 3 Determination method of detection kit

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

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

[0060] (2) Take the operation of Japan East Asia CA530 coagulometer as an example, according to the instrument description, set the analysis program: the measurement wavelength is 405nm;

[0061] Take 5μl of plasma sample, add 120μl of physiological saline to dilute, the dilution ratio is 1:25, incubate at 37℃ for 30 seconds, take 50μl of diluted sample, add 60μl of R1 reagent and incubate at 37℃ for 90 seconds, then add 60μl of R2 reagent at 37℃ Incubate and measure the absorbance difference (△OD) at the 10th second and the 40th second. The instrument automatically dilutes the calibrator into 5 stand...

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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 diagnostics, in particular to a kit for detecting antithrombin III by a chromogenic substrate method. Background technique [0002] Antithrombin III (AT-III) is a heparin-dependent serine protease inhibitor and an important anticoagulant factor, which bears 60%~70% of antithrombin in plasma Activity plays an important role in maintaining the balance of physiological blood coagulation and anticoagulation. AT-III is synthesized by liver, vascular endothelial cells and megakaryocytes, with a molecular weight of about 60,000 Da, belongs to α2-globulin, and its gene is located on chromosome 1 (1p23). [0003] In the process of thrombosis, AT-III is a very important regulator. Under the catalysis of heparin, it can be combined with thrombin or coagulation factors IXa, Xa, XIa, XIIa, plasmin and other serine proteases in a ratio of 1:1 A complex is formed, thereby deactivating these enzymes and exerting an ant...

Claims

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Application Information

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IPC IPC(8): C12Q1/56C12P19/12
Inventor 谢永华
Owner SHANGHAI SUNBIO TECH
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