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Kit for high-sensitivity detection on proteoglycan and preparation method of kit

A proteoglycan, high-sensitivity technology, applied in the fields of biological analysis and medical detection, can solve the problems of different sulfation degrees and coupling sites, complex proteoglycan composition, and low detection sensitivity

Active Publication Date: 2016-05-25
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Due to the different molecular weights and structures of proteoglycan core proteins, as well as the number, type, length, degree of sulfation and coupling sites of covalently coupled GAG chains, the composition of proteoglycans is complex and the structures are diverse. Its detection poses many difficulties
In the detection of GPC3, the most commonly used detection method is the sandwich ELISA method, but its detection sensitivity is low, and only about 50% of HCC patients can detect the obvious presence of GPC3 in the serum of HCC patients; The adsorption coating and the combination of the second antibody and the antigen need to be incubated overnight, and it takes three days to complete an experiment, which takes a long time, which greatly limits the application of this method and brings difficulties to the detection of GPC3

Method used

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  • Kit for high-sensitivity detection on proteoglycan and preparation method of kit
  • Kit for high-sensitivity detection on proteoglycan and preparation method of kit
  • Kit for high-sensitivity detection on proteoglycan and preparation method of kit

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Experimental program
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Effect test

Embodiment 1

[0073] A kit for high-sensitivity detection of proteoglycan GPC3, comprising:

[0074] Carboxygraphene coupled to monoclonal antibody 1, said monoclonal antibody 1 is an antibody prepared after immunization with the 70 amino acid sequence fragments at the carboxyl end of the core protein of proteoglycan; said coupled monoclonal antibody 1 The concentration of carboxygraphene is 10 μg / mL;

[0075] The carboxyl graphene of coupling monoclonal antibody 2, described monoclonal antibody 2 is the antibody that the amino terminal 510 amino acid sequence fragments of the core protein of proteoglycan are prepared after antigen immunization; Said coupling monoclonal antibody 2 The concentration of carboxygraphene is 10 μg / mL;

[0076] Highly positively charged green fluorescent protein (ScGFP); the concentration of highly positively charged green fluorescent protein is 0.005 μg / μL;

[0077] Buffer: a solution containing a concentration of 10mM Tris-HCl and a concentration of 100mMNaCl...

Embodiment 2

[0100] Embodiment 2, optimization of reaction conditions

[0101] Effect of reagent addition sequence on GPC3 detection

[0102] Add 0.05-0.2 μg of carboxygraphene coupled with αGCN, 0.05-0.2 μg of carboxygraphene coupled with αGCC, 0.01-0.5 μg of ScGFP, and 30.001-0.005 ng of GPC into the 40 μL reaction system of the microwell plate in different order, Mix well, and place at room temperature in the dark for 15 minutes. After incubation, measure the fluorescence intensity (Ex: 470nm, Em: 509nm).

[0103] Among them: Group A is carboxygraphene coupled with αGCN, GPC3 and ScGFP mixed and then added with carboxygraphene coupled with αGCC; Group B is two kinds of carboxygraphene coupled with αGCN and αGCC respectively mixed with GPC3 and then added with ScGFP; C Group D is two kinds of carboxygraphene coupled with αGCN and αGCC, mixed with ScGFP and then added with GPC3; group D is mixed with carboxygraphene coupled with αGCC, GPC3 and ScGFP, and then added with carboxygraphene c...

Embodiment 3

[0122] Embodiment 3, the detection of GPC3 in buffer solution

[0123]Add 0.05-0.2 μg of carboxygraphene coupled with αGCN and 30-0.04 ng of GPC to 40 μL of 10 mMTris-HCl and 100 mM NaCl solution in a microwell plate, mix well, and place at room temperature for 5 minutes; then add ScGFP 0.01-0.5 μg, mix well, and store at room temperature Place it in the dark for 15 minutes; finally add 0.05-0.2 μg of carboxygraphene coupled with αGCC, mix well, and place it in the dark at room temperature for 10 minutes. After the incubation, measure the fluorescence intensity (Ex: 470nm, Em: 509nm). The schematic diagram of proteoglycan GPC3 detection process is as follows: Figure 9 shown.

[0124] The results show that with the increase of GPC3 concentration, the fluorescence quenching intensity mediated by it also gradually increases, and the detection sensitivity can reach below 10pg / mL (such as figure 2 ), much higher than the sandwich ELISA method.

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Abstract

The invention relates to a kit for high-sensitivity detection on proteoglycan and a preparation method of the kit. The kit comprises carboxyl graphene coupled with a monoclonal antibody and / or carboxyl graphene coupled with a polyclonal antibody, fluorescent proteins with high-positive charges, and a buffering solution. The invention further relates to a preparation method of the kit. According to the kit for high-sensitivity detection on the proteoglycan, an elisa plate is not needed to be adsorbed and wrapped by an antibody, and such complicated steps of washing antigen and antibody combination and the like are eliminated; and furthermore, quick detection and analysis on target proteoglycan in a homogeneous system can be realized, and the sensitivity is high.

Description

technical field [0001] The invention relates to a kit for high-sensitivity detection of proteoglycans and a preparation method thereof, belonging to the technical fields of biological analysis and medical detection. Background technique [0002] Proteoglycan (proteoglycan, PG) is a kind of complex biomacromolecule, and one or more glycosaminoglycan (glycosaminoglycan, GAG) chains are covalently coupled to the core protein. GAG is a linear polysaccharide composed of repeating disaccharide units, which are mainly divided into four categories: hyaluronic acid (HA), chondroitin sulfate (CS) / dermatan sulfate (DS), keratan sulfate (KS) and heparin ( Hep) / heparan sulfate (HS). Among them, hyaluronic acid does not combine with core protein to form proteoglycan. Proteoglycans contain some N- or O-linked oligosaccharide chains in addition to glycosaminoglycan chains. [0003] PG is widely distributed in the extracellular matrix and is also present on the cell surface as well as in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577
CPCG01N33/577G01N33/68
Inventor 李福川韩乃寒王文爽蔡晓娟
Owner SHANDONG UNIV
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