Kit for determining heparin

A technology for heparin determination and kit, which is applied in biological tests, measuring devices, material inspection products, etc., can solve the problems of reducing the simplicity of the heparin determination kit, and achieve the effects of simple operation, high sensitivity, and cost reduction.

Inactive Publication Date: 2018-02-23
SHANGHAI SUNBIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This reduces the simplicity of the heparin assay kit (chromogenic substrate method) to a certain extent

Method used

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  • Kit for determining heparin
  • Kit for determining heparin
  • Kit for determining heparin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Composition and preparation method of embodiment 1 heparin assay kit

[0037] R1 reagent is mainly prepared from the following raw materials: porcine FXa concentration is 1.25U / ml, Tris is 50mM, BSA is 6.64mg / ml, polyethylene glycol-6000 is 10mg / ml and mannitol is 100mM, adjusted with 6M HCl pH to 8.4, lyophilized in 1ml / vial aliquots.

[0038] The R2 reagent was prepared from the following reagents: the chromogenic substrate Suc-Ile-Glu-(γ-Piperidyl)-Gly-Arg-pNA·HCl was 3.0 mg / ml, the acharan sulfate lyase was 1.0 U / ml, and Tris was 50mM, 0.02g / L dextran sulfate, 10mg / ml polyethylene glycol-6000 and 100mM mannitol, adjust the pH to 7.5 with 6M HCl, and freeze-dry in 1ml / bottle.

Embodiment 2

[0039] The assay method of embodiment 2 detection kits

[0040] (1) Reconstitute the R1 reagent and R2 reagent obtained in Example 1:

[0041] Reconstitute each bottle of R1 reagent with 5ml of distilled water, and each bottle of R2 reagent with 2ml of distilled water.

[0042] (2) Taking the operation of BioTek ELx800 microplate reader (end point method) as an example, set the analysis program according to the instrument instructions: the measurement wavelength is 405nm;

[0043] Firstly, dilute the heparin with known potency to 100U / ml heparin with normal saline. Take 10 μl of 100 U / ml heparin, add 990 μl of human plasma standard substance to dilute, and obtain 1.0 U / ml heparin standard substance. Continue to dilute the 1.0U / ml heparin standard with the human plasma standard, prepare 5 standard samples of 0.0, 0.2, 0.5, 0.8, 1.0U / ml heparin, and incubate at 37°C for 30 seconds. Take 50 μl of the diluted sample, add 50 μl of R2 reagent and incubate at 37°C for 60 seconds, ...

Embodiment 3

[0048] Embodiment 3 Analytical performance evaluation of the kit of the present invention

[0049] (1) Sensitivity

[0050] With the heparin standard product of 0.0U / ml as blank sample, use the test kit of the present invention obtained in Example 1, the detection method described in Example 2, repeat the measurement 20 times, and calculate the OD of this sample 405nm average and standard deviation (SD), the blank mean minus twice the standard deviation was substituted into the calibration curve equation to calculate the lowest detection limit, and the results are shown in Table 2.

[0051] Table 2 Minimum detection limit analysis

[0052]

[0053] The results in Table 2 show that the minimum detection limit of the kit of the present invention for detecting heparin can reach 0.02 U / ml, which is lower than that of the commercially available heparin detection kit (chromogenic substrate method, BIOPHEN Heparin kit of Aniara Company in France, article number: 221006) instruc...

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Abstract

The invention belongs to the field of medical in vitro diagnosis and specifically relates to a kit for determining heparin. The kit comprises pig FXa, acharan sulfate lyase and chromogenic substrate.The FXa of the kit provided by the invention is prepared from pig plasma, to solve the problem of the plasma source, the preparation is suitable for the national condition of China, the preparation isconvenient and the cost is lowered. The kit provided by the invention adopts a standard curve for detecting various heparin drugs; multiple standard curves for different heparin drugs are not required; the operation is simple and convenient; the dilution is not required by the detection for the plasma sample through the kit provided by the invention; the detection result is high in sensitivity, high in stability and high in accuracy.

Description

technical field [0001] The invention belongs to the field of medical in vitro diagnosis, and specifically relates to a heparin assay kit, in particular to a heparin assay kit for detecting heparin activity by a chromogenic substrate method (one-step method). Background technique [0002] Heparin is an acidic mucopolysaccharide synthesized by mast cells distributed in the intestinal mucosa. There is very little content in the blood of normal people, and its anticoagulant effect is very small under physiological conditions. It can play an anticoagulant role by accelerating antithrombin and HCⅡ, and also has an effect on TFPI and protein C systems. Natural heparin is heterogeneous, and its molecular weight is between 3-57kD (Wang Zhenyi. Thrombosis and Hemostasis Basic Theory and Clinic [M]. Third Edition. Shanghai: Shanghai Basic Science Publishing House, 2004: 119-119). Clinically, heparin is widely used as an anticoagulant drug, mainly used in the treatment of thrombotic d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/86G01N33/535
CPCG01N33/535G01N33/86G01N2400/40
Inventor 谢永华
Owner SHANGHAI SUNBIO TECH
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