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A fluorescent chip quantitative detection kit

A quantitative detection and kit technology, applied in the field of protein detection, can solve the problems of high instrument cost, large instrument, large sample size, etc., and achieve the effect of high sensitivity

Active Publication Date: 2022-07-26
ZHEDA DIXUN BIO GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the methods for detecting cytokines include flow cytometry and enzyme-linked immunoassay. The cost of flow cytometry is high, and the cost of equipment is high.
At present, most of the allergy-specific IgE tests on the market are qualitative, and quantitative ones require larger instruments, and the experiment takes a long time and requires a large sample size.
[0008] Cytokines, allergen protein-specific IgE, IgG (including IgG4), IgA and other items are items with many indicators. At present, the methods that can be detected on the market have large instruments, high detection costs, or long experiment time and large sample size. And other issues

Method used

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  • A fluorescent chip quantitative detection kit
  • A fluorescent chip quantitative detection kit
  • A fluorescent chip quantitative detection kit

Examples

Experimental program
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Effect test

preparation example Construction

[0042] In the present invention, the preparation method of the detection plate immobilized with the cytokine-specific monoclonal antibody to be tested comprises the following steps:

[0043] Use streptavidin to coat each detection site of the detection plate to obtain a coated detection plate; label the cytokine-specific monoclonal antibodies to be tested with biotin respectively to obtain a biotin-labeled test plate to be tested Cytokine-specific monoclonal antibody; the biotin-labeled cytokine-specific monoclonal antibody to be tested is respectively coupled to different detection sites on the detection plate to obtain a detection plate with the cytokine-specific monoclonal antibody to be tested immobilized.

[0044] In the present invention, streptavidin is used to coat each detection site of the detection plate to obtain a coated detection plate. The present invention does not specifically limit the coating method, and conventional streptavidin coating methods well known t...

Embodiment 1

[0084] Preparation of the kit of the invention

[0085] 1. Polystyrene detection plate immobilized cytokine-specific monoclonal antibodies

[0086] A. Prepare commercially available streptavidin with 0.01M, pH 7.4 PBS to a suitable concentration (eg: 0.1~2mg / mL), add 0.5~2uL to each detection site in the reaction tank of the detection plate, The reaction was allowed to stand at 4°C overnight (more than 16h);

[0087] B. Add 0.5-1.5mL, 0.01M, pH7.4 PBS containing 0.05% Tween 20 to wash the reaction tank of the detection plate once and pat dry.

[0088] C. Add 0.5-1.5mL, 0.01M, pH7.4 PBS containing 2% BSA to each reaction tank to block, and let stand at 4°C for overnight reaction (more than 16h);

[0089] D. Add 0.5-1.5mL, 0.01M, pH7.4 PBS containing 0.05% Tween 20 to wash the detection plate once and pat dry.

[0090] E. Prepare the biotin-labeled cytokine monoclonal antibody with 0.01M PBS, pH 7.4 to a suitable concentration (eg: IL-1beta: 0.1-7.0 mg / mL; IL-2: 2.0-5.0 mg / mL...

Embodiment 2

[0105] Dual signal amplification system using fluorescence method plus biological method (biotin-streptavidin method)

[0106] For the preparation of the detection kit, see Example 1

[0107] The detection method is as follows:

[0108] (1) Take the detection plate solidified with different cytokine monoclonal antibodies, and place it horizontally on a special plate rack at room temperature for standby;

[0109] (2) Add 200-400 μL of serum or plasma to each reaction tank, place the plate rack on a mixer, and incubate at room temperature for 30-60 minutes;

[0110] (3) Rinse the liquid reaction tank with the washing liquid, and repeat the cleaning for 3 to 5 times, each time for 10 to 30 s;

[0111] (4) Dilute 200-400 μL of fluorescent microsphere-conjugated mixed paired antibody working solution with PBS 1:10-1000 (volume ratio), add it to each reaction tank, and incubate at room temperature on a mixer for 30-60 min;

[0112] (5) Rinse the liquid reaction tank with the wash...

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Abstract

The invention relates to a fluorescent chip quantitative detection kit, which belongs to the technical field of protein detection. The kit of the present invention includes a detection plate and a detection antibody coupled to fluorescent microspheres; the detection plate is provided with a plurality of reaction tanks, the reaction tanks are provided with openings, and the inner bottom surface of the reaction tank is along the length of the reaction tank A plurality of detection sites are arranged side by side and spaced in the direction. The kit of the invention can detect cytokines or allergen protein-specific IgE, IgG and IgA with high sensitivity, and can rapidly and quantitatively detect the concentration of cytokines in human serum or plasma, and can detect more than a dozen cytokines at one time. It can rapidly and quantitatively detect the concentration of allergen protein-specific antibodies IgE, IgG and IgA in human serum or plasma, and can screen dozens of allergens at one time.

Description

technical field [0001] The invention relates to the technical field of protein detection, in particular to a high-sensitivity, fluorescent chip quantitative detection kit for the levels of various cytokines and allergen-specific IgE, IgG and IgA concentrations in human serum or plasma, IgG including IgG4. Background technique [0002] The term cytokine storm (hypercytokineemia) was first proposed in 1993 as the pathogenesis of graft-versus-host disease (GVHD). The use of the term in infectious disease research began in the early 2000s in reports of cytomegalovirus, hemophagocytic lymphohistiocytosis, influenza virus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), etc. used. Cytokine storm is an important cause of acute respiratory distress syndrome (ARDS) and multiple organ failure, and its concentration correlates with disease severity and prognosis. [0003] Cytokines are low-molecular-weight soluble proteins produced by a variety of cells induced by im...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/58G01N33/545G01N33/68G01N33/577G01N33/547
CPCG01N33/582G01N33/587G01N33/545G01N33/6863G01N33/6866G01N33/6869G01N33/6854G01N33/6893G01N33/577G01N33/54353G01N2800/24G01N2800/245G01N2800/26G01N2800/60G01N2800/52B01L3/50855B01L2200/16G01N33/54387Y02A50/30
Inventor 吴周杰刘奕吴善东沈华浩陈姗姗吴绍长王溢飞朱明芝王美杰陈初含
Owner ZHEDA DIXUN BIO GENE ENG
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