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Kit as well as preparation method and application thereof

A kit and inhibitor technology, applied in the field of medical diagnosis, can solve problems such as difficulty in obtaining, increasing market demand, and high price

Pending Publication Date: 2022-03-25
深圳市雅为泓源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are only a small number of foreign and domestic manufacturers with this kit in the market, and they are all expensive and difficult to obtain, and there are few commercial kits; with the clinical promotion, tissue plasminogen activator-plasminogen activator inhibitor-1 Complex (t-PAI-C) commercial assay kit market demand is also increasing sharply

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  • Kit as well as preparation method and application thereof
  • Kit as well as preparation method and application thereof
  • Kit as well as preparation method and application thereof

Examples

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preparation example Construction

[0052] The present invention further proposes a preparation method of the kit as described above, comprising the following steps:

[0053] S10. Coupling the biotin to the coating antibody, removing excess biotin, and then diluting with the first buffer solution to obtain the first working solution.

[0054] For specific operations, the following methods can be used:

[0055] Take 60 μg of coated antibody, and dilute the antibody to a final concentration of 2 mg / mL with biotinylated activation buffer (the biotinylated activation buffer is 50 mM phosphate buffer, containing 50 mM disodium hydrogen phosphate and a mass fraction of 0.9 %NaCl, pH value is 8.0), weighed 0.3mg of biotin, and added 300μL of DMSO (dimethyl sulfoxide) to prepare a 10mg / mL biotin solution, and added 4-10μL of biotin solution to the coating antibody solution Incubate at 22°C in the dark for 0.5-3 hours, purify with a desalting column to remove excess unreacted biotin, and dilute to 50-500 mL with biotiny...

Embodiment 1

[0084] Example 1 Kit

[0085] The first working solution: biotinylated coating antibody (coating antibody: the monoclonal antibody whose article number is YM0213, Shenzhen Yawei Century Technology Co., Ltd.), the first buffer solution [50mM disodium hydrogen phosphate, 0.9% NaCl (w / w), 1% BSA (w / w), 0.5mM CaCl 2 , 0.05% Triton X-100 (w / w), 0.05% Tween-20 (w / w), 1% glycine (w / w)], wherein the pH value of the first buffer is 7.2;

[0086] The second working solution: superparamagnetic particles coated with streptavidin, the second buffer [50mM Tris, 0.9% NaCl (w / w), 0.05% Tween-20 (w / w), 0.05% Triton X-100 (w / w), 0.05% Proclin300 (w / w)], wherein the pH value of the second buffer is 7.2;

[0087] The third working solution: labeled antibody labeled with enzyme (labeled antibody: monoclonal antibody with product number YM0212, Shenzhen Yawei Century Technology Co., Ltd.), the third buffer solution [0.05M MES, 0.15M NaCl, 0.1% Tween- 20(w / w), 0.05% Proclin300(w / w), 1% BSA(w / w),...

Embodiment 2

[0090] The preparation method of embodiment 2 kit

[0091] The material preparation kit according to embodiment 1 may further comprise the steps:

[0092] 1. Preparation of various buffer solutions

[0093] Second buffer: 50mM Tris, 0.9% NaCl (w / w), 0.05% Tween-20 (w / w), 0.05% TritonX-100 (w / w), 0.05% Proclin300 (w / w), pH is 7.2;

[0094] The third buffer: 0.05M MES, 0.15M NaCl, 0.1% Tween-20 (w / w), 0.05% Proclin300 (w / w), 1% BSA (w / w), 1% glycine (w / w) , 10% Glycerol (w / w), 5mM MgCl 2 , 0.1 mM ZnCl 2 , the pH value is 6.0;

[0095] First buffer: 50 mM disodium phosphate, 0.9% NaCl (w / w), 1% BSA (w / w), 0.5 mM CaCl 2 , 0.05% Triton X-100 (w / w), 0.05% Tween-20 (w / w), 1% glycine (w / w), pH value is 7.2;

[0096] Biotinylation activation buffer: 50mM phosphate buffer, containing 50mM disodium hydrogen phosphate, and 0.9% NaCl (w / w), pH 8.0;

[0097] Enzyme blocking solution: 0.1M disodium hydrogen phosphate, 0.15mM NaCl, 50mM hydroxylamine hydrochloride, pH value is 7.2.

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Abstract

The invention discloses a kit as well as a preparation method and application thereof, and relates to the technical field of medical diagnosis, the kit is used for detecting the content of a tissue plasminogen activator-plasminogen activator inhibitors 1 compound, the kit comprises a first working solution, a second working solution and a third working solution, the first working solution comprises a biotinylation coated antibody and a first buffer solution, and the second working solution comprises a second buffer solution and a third buffer solution; the biotinylated coated antibody is obtained by coupling biotin and a coated antibody; the second working solution comprises superparamagnetic particles coated with streptavidin and a second buffer solution; the third working solution comprises a labeled antibody labeled with an enzyme and a third buffer solution, and the labeled antibody labeled with the enzyme is obtained by coupling the enzyme activated by carbodiimide and N-hydroxysuccinimide with the labeled antibody; a luminescent substrate; and a cleaning solution. When the kit provided by the invention is used for detecting the content of the plasminogen activator-plasminogen activator inhibitor 1 compound in the tissue, the accuracy is high, and the stability is good.

Description

technical field [0001] The invention relates to the technical field of medical diagnosis, in particular to a kit and its preparation method and application. Background technique [0002] Tissue plasminogen activator inhibitor (PAI-1) is a physiological inhibitor of tissue plasminogen activator (t-PA). The activity of t-PA to activate plasminogen is inhibited, thereby inhibiting the degradation of fibrin. When blood coagulation is activated, fibrin formation can activate tissue plasminogen activator (t-PA) synthesized in vascular endothelial cells, and the activated t-PA further activates the conversion of plasminogen into plasmin, which degrades Fibrin produces fibrin degradation products. The concentration of PAI-1 in plasma is 5 times that of t-PA, much higher than t-PA, so it can be considered that almost all t-PA released into the blood forms a complex with PAI-1. The level of t-PAI-C is positively correlated with the concentration of t-PA and the degree of vascular e...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N33/543
CPCG01N33/573G01N33/543
Inventor 杨华程晓东邓站胜李振江邹小宇
Owner 深圳市雅为泓源生物科技有限公司
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