Human brain glioma marker glial fibrillary acidic protein (GFAP) autoantibody and applications thereof

A technology of human glioma and autoantibodies, which is applied in the fields of biotechnology and medicine, can solve the problems of high cost of imaging diagnosis technology and the inability to be widely used

Inactive Publication Date: 2012-07-04
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing imaging diagnostic techniques cannot be widely used due to their high cost, so the development of relatively cheap early detection methods for glioma based on clinical blood samples has become a hot spot for those skilled in the art

Method used

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  • Human brain glioma marker glial fibrillary acidic protein (GFAP) autoantibody and applications thereof
  • Human brain glioma marker glial fibrillary acidic protein (GFAP) autoantibody and applications thereof
  • Human brain glioma marker glial fibrillary acidic protein (GFAP) autoantibody and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Serum collection and its clinical information

[0032] Serum samples of glioma patients of different grades were collected from Shanghai Huashan Hospital, and 41 healthy people of corresponding age and sex from Huashan Hospital were used as normal controls. All samples were stored in a -80°C freezer. Patient serum samples were classified into astrocytoma, anaplastic astrocytoma, and glioblastoma multiforme according to their respective histopathological analysis. Using proteomics methods, twenty glioblastoma multiforme patient serum samples and their corresponding normal human serum samples were used to detect and screen for autoantibodies. 81 serum samples were used to detect and confirm the presence of GFAP autoantibodies (this research has been approved by the Ethics Committee of Huashan Hospital and Biomedical Research Institute affiliated to Fudan University).

Embodiment 2

[0033] Example 2: 2D-Western analysis

[0034]U251 brains collected by centrifugation were lysed using urea buffer (8M urea, 2.5M thiourea, 65mM DTT, 4% (w / v) CHAPS, 0.5% (v / v) Biolytes pH 3-10, protease inhibitor cocktail) Glioma cells were quantified using RC DC Protein Assay Kit (Bio-Rad), and 30 micrograms of protein were diluted in rehydration buffer (7M urea, 2M thiourea, 50mM DTT, 4% (w / v) CHAPS, 0.2 % (v / v) Biolytes pH 3-10), and loaded in IPG strips. Isoelectric focusing was performed using Protean IEF Cell (BioRad), followed by SDS-PAGE electrophoresis. Separated proteins were visualized by silver staining or transferred to PVDF membrane overnight with a 100 mA current. The same protein was run on two gels in parallel, one with patient serum for western blotting, and the other for western blotting with normal human serum. After blocking for two hours in TBST containing 4% BSA, the two membranes were incubated in 1:200 dilution of patient and corresponding normal h...

Embodiment 3

[0035] Embodiment 3: image analysis and protein identification

[0036] LAS-3000 and supporting software Gauge V3.0 software (Fuji, Japan) were used to analyze and identify the difference of immune reaction spots on the membrane incubated with patient and corresponding normal human serum. The corresponding position of each protein spot was confirmed by comparing the pictures of silver-stained 2D gel and western blot. Proteins that only appear in human serum were spot-cut, decolorized, enzymatically hydrolyzed and freeze-dried. The process is to destain the excised strips in 50mM NH4HCO3, 50% acetonitrile, then add 10mM DTT (Amersco, Solon, Ohio) at 56°C for reduction, and incubate in the dark by adding 55mM iodoacetamide (Bio-Rad) Alkylation. The gel pieces were dehydrated with acetonitrile and air dried. 25 mM ammonium bicarbonate containing 0.01 μg / ul trypsin (Promega) was added to carry out in-gel enzymatic hydrolysis at 37 degrees for 16 hours, and then the peptide was ...

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Abstract

The invention belongs to the fields of biotechnology and medicine, relates to a human brain glioma marker, and in particular relates to a detection method and applications of a glial fibrillary acidic protein (GFAP) autoantibody capable of distinguishing the human brain glioma. The purified GFAP is used to perform enzyme-linked immunosorbent assay (ELISA) analysis, the levels of GFAP autoantibodies in the serum of normal people and in the serum of patients with brain glioma at different levels are detected, the results show that the level of GFAP autoantibodies in normal serum is minimum and the level increases with the increase of the level of glioma; linear regression shows that the correlation of the level of GFAP autoantibodies and the level of the brain glioma is significant; and the two-tailed student t-test shows that compared with normal control, the levels of the GFAP autoantibodies of the third grade brain glioma and the fourth grade brain glioma are both significant. Experiments show that the GFAP autoantibodies can be used as the marker of the human brain glioma in the early detection and diagnosis of the brain glioma.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine. The invention relates to markers of human brain glioma, in particular to a protein marker GFAP (glial fibrillary acidic protein, GFAP) autoantibody capable of distinguishing human brain glioma, a detection method and application thereof. Background technique [0002] Glioma is a tumor originating from glial cells, which is the most common brain tumor. Existing diagnostic imaging techniques cannot be widely used due to their high cost. Therefore, the development of relatively cheap early detection methods for glioma based on clinical blood samples has become a hot spot for those skilled in the art. Autoantibodies are antibodies produced by the immune system against collective self-proteins, cells, tissues or organs, which are closely related to autoimmune diseases. At the same time, related studies have also identified antibody production caused by tumor-associated autoantigens in many...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/574
Inventor 施前危平张伟杨柳松
Owner FUDAN UNIV
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