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Method for detecting core fucosylation level of serum IgG

A technology of fucosylation and fucosylation, which is applied in the field of proteins and can solve the problems of reduced ligand affinity and abnormal activation.

Inactive Publication Date: 2018-07-10
DALIAN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Our previous study found that loss of core fucosylation on transcriptional growth factor beta 1 (TGFβ-1) receptors and or epidermal growth factor (EGF) receptors exhibited marked activation abnormalities attributed to ligand Decreased affinity for receptors

Method used

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  • Method for detecting core fucosylation level of serum IgG
  • Method for detecting core fucosylation level of serum IgG
  • Method for detecting core fucosylation level of serum IgG

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Detection and analysis of IgG core fucosylation level in normal control group and autoimmune disease group

[0044] 1. Sample collection

[0045] Serum samples were collected from 37 cases who came to Dalian Central Hospital for treatment from September 2015 to September 2016 and were diagnosed with autoimmune diseases, including 11 males and 26 females, with an average age of 51.6±13.2 years. In addition, 20 healthy subjects were selected as the normal control group, with an average age of 51.2±10.3 years. Extract fasting whole blood, let stand for 30 minutes, 3000rpm, 10min. Aspirate the upper layer of serum and store it in a -80°C refrigerator for later use. Antinuclear antibody titers in serum of patients with autoimmune diseases (shown in Table 1).

[0046] Table 1 Serum samples from patients with autoimmune diseases

[0047]

[0048]

[0049]

[0050] 2. Prokaryotic expression of AOL lectin that specifically binds to core fucose, for detection of core...

Embodiment 2

[0085] Detection of serum in patients with autoimmune diseases by sandwich method ELISA

[0086] 1 coat

[0087] Dilute the coated protein G with pH 9.6 carbonate buffer to reach a concentration of 5-20 μg / ml, 50 μl per well to coat the microtiter plate, and coat overnight at 4°C.

[0088] 2 wash plates

[0089] Dry the coating solution, wash with PBST, 200 μl / well, and dry the washing solution after 30 seconds each time, repeat three times.

[0090] 3 closed

[0091] Add 200 μl / well of blocking solution, and incubate at 37°C for 30 minutes in a humid chamber.

[0092] 4 Add the serum sample to be tested

[0093] Add normal human serum samples and patient serum samples diluted 1:10-1:100 times. React at 37°C for 1h. The negative control was PBS; the positive control was serum samples from confirmed patients.

[0094] 5 wash plate

[0095] Wash with PBST, 200 μl / well, shake off the washing solution after each 30s, repeat three times.

[0096] 6 primary antibody reactio...

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Abstract

The invention discloses a method for detecting the core fucosylation level of serum IgG. The method comprises a step of specifically identifying core fucosyl groups in the serum IgG by using lectin, but not comprises a step of sampling from an organism, and a serum sample is an in vitro sample. The lectin is an Aspergillus oryzae lectin AOL protein (domain) which can specifically identify the corefucosyl groups, and is obtained by prokaryotic expression, and the amino acid sequence of the lectin is represented by SEQ ID NO. 1; and the lectin is encoded by an FleA gene fragment represented bySEQ ID NO. 2. The method has potential application values in the screening of autoimmune diseases and the detection of the autoimmune diseases and provides a potential therapeutic way for possible immune regulation intervention after being combined with clinical anti-nuclear antibody titer detection.

Description

technical field [0001] The invention belongs to the technical field of proteins and relates to the detection of glycoproteins, in particular to the detection of serum IgG core fucosylation. Background technique [0002] Autoimmune diseases refer to diseases caused by the body's immune response to self-antigens, resulting in damage to its own tissues. It has the following characteristics: 1) The patient has obvious familial tendencies; 2) There are high titers of autoantibodies in the blood; 3) Autoimmune diseases often present recurrent and chronic protracted processes; 4) Autoimmune diseases are often accompanied by Abnormal function and complications of vital organs such as liver, kidney, and intestines. Currently there is no cure but only symptomatic treatment. If autoimmune diseases can be detected at an early stage, monitored for development and changes, and treated in time, the occurrence of complications can be delayed. [0003] At present, the screening methods fo...

Claims

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Application Information

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IPC IPC(8): G01N33/535C12N15/70
CPCC12N15/70G01N33/535
Inventor 李文哲李志李明梁伟李雪滢
Owner DALIAN MEDICAL UNIVERSITY
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