Method for multiply detecting carbohydrate chain structure of glycoprotein through antibody-assisted lectin liquid-phase suspension chip

A suspension chip, multiple detection technology, applied in the biological field, to achieve the effects of improving detection throughput, good reproducibility, and good clinical application prospects

Inactive Publication Date: 2014-03-19
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to make lectin chip technology better applied to clinical sample glycosylation detection and glycosylation differential screening, the detection throughput of lectin chip technology needs to be improved urgently

Method used

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  • Method for multiply detecting carbohydrate chain structure of glycoprotein through antibody-assisted lectin liquid-phase suspension chip
  • Method for multiply detecting carbohydrate chain structure of glycoprotein through antibody-assisted lectin liquid-phase suspension chip
  • Method for multiply detecting carbohydrate chain structure of glycoprotein through antibody-assisted lectin liquid-phase suspension chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 The minimum detection limit and linear range experiment of antibody auxiliary lectin liquid phase suspension chip

[0025] Use a series of concentrations of haptoglobin (Hp) solution and incubate with 2.5 μL magnetic beads overnight at 4°C. After washing, add IgG to block at room temperature; after washing again, add biotinylated antibody solution, incubate at room temperature for 1 hour, wash and Add the secondary antibody reaction solution; after incubating at room temperature for 30 minutes, use the Bioplex suspension chip detection system to collect the signal; the sugar chain structure is similar to haptoglobin (Hp), but cannot be recognized by the haptoglobin (Hp) antibody Transferrin (Tf) was used as a negative control; when the net signal was greater than 3 times the standard deviation, and the signal-to-noise ratio was greater than 1.5 times the minimum glycoprotein concentration was the detection limit. The result is as figure 2 A. figure 2 B. ...

Embodiment 2

[0026] Example 2 Specificity Experiment of Antibody-assisted Lectin Liquid Phase Suspension Chip Multiple Detection Method

[0027] To 3ng / mL haptoglobin (Hp) solution, add 2.5 μL each of RCA120 and SNA-I coupled magnetic beads, incubate overnight at 4°C, add IgG to block at room temperature after washing; add biotinylated Haptoglobin (Hp) antibody solution, after incubation at room temperature for 1 hour, wash and add secondary antibody reaction solution; after incubation at room temperature for 30 minutes, use the Bioplex suspension chip detection system to collect signals; sugar chain structure, but transferrin (Tf) that cannot be recognized by the haptoglobin (Hp) antibody was used as a negative control; the results were as follows image 3 As shown in A, the signal intensity of the multiple detection method is consistent with that of the single detection method, indicating that the multiple detection method can specifically identify different sugar chain structures on the...

Embodiment 3

[0028] Example 3 Dose-increasing experiment of antibody-assisted lectin liquid-phase suspension chip multiple detection method

[0029] Prepare different concentrations of haptoglobin (Hp) solutions, add 2.5 μL of RCA120 and SNA-I-coupled magnetic beads to each sample, incubate overnight at 4°C, add IgG to block at room temperature after washing; after washing, add Biotinylated haptoglobin (Hp) antibody solution was incubated at room temperature for 1 hour, washed and added to the secondary antibody reaction solution; after 30 minutes of room temperature incubation, the signal was collected with the Bioplex suspension chip detection system; with haptoglobin ( Hp) has a similar sugar chain structure, but transferrin (Tf), which cannot be recognized by the haptoglobin (Hp) antibody, was used as a negative control; the results were as follows image 3 In B, the lectin signal increases with increasing glycoprotein concentration in the multiplex assay.

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Abstract

The invention belongs to the technical field of biology and relates to a method for multiply detecting a carbohydrate chain structure of glycoprotein through an antibody-assisted lectin liquid-phase suspension chip. According to the method, lectins are coupled to magnetic beads marked with different dyes, the magnetic beads which are coupled with different lectins are identified through a Bioplex detection system, and different glycoforms on the same protein can be simultaneously detected by adding the magnetic beads which are coupled with different lectins and have different serial numbers into a reaction, so that the detection flux of the carbohydrate chain structure is remarkably improved. According to the method, the carbohydrate chain structure on the glycoprotein can be quickly and specifically detected with high flux. The method can be further applied to detection of a clinical biological marker and provides an efficient way for research on disease glycomics.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a new method for multiple detection of glycoprotein sugar chain structure by an antibody-assisted lectin liquid-phase suspension chip; in particular, it relates to the preparation of an antibody-assisted lectin liquid-phase suspension chip and the antibody-assisted lectin liquid The phase chip performs multiple detection of the glycoforms on haptoglobin (Hp); this method can perform multiple detection of the sugar chain structure on the glycoprotein, and improves the throughput of the lectin chip technology. Background technique [0002] Glycosylation of eukaryotic proteins is a pervasive, crucial, yet complex post-translational modification; changes in glycosylation structure are a widespread hallmark in cancer. Some sugars located at the end of the sugar chain, including sialylated Lewisx (sLe x ), sialylated Tn (sTn), Globo H, Lewis y (Le y ) and polysialic acid are overex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531
CPCG01N33/68G01N33/54326G01N2400/02G01N2570/00
Inventor 汪泓李宏余红秀杨芃原
Owner FUDAN UNIV
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