Epitope for sicca syndrome and application thereof

A technology for Sjogren's syndrome and antigenic epitopes, which is applied in the field of immunological diagnosis, and can solve the problems of self-antigen detection antibodies and unclear pathogenesis

Inactive Publication Date: 2012-11-28
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The appearance of a large number of autoantibodies is one of the important characteristics of autoimmune diseases, but because its pathogenesis is still unclear, it is difficult to directly use autoantigens to detect antibodies

Method used

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  • Epitope for sicca syndrome and application thereof
  • Epitope for sicca syndrome and application thereof
  • Epitope for sicca syndrome and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 SS patient serum total IgG preparation and purification

[0021] Take an appropriate amount of Protein A resin suspension (product of GE Company), put it into the chromatography column, and wash the equilibrium chromatography column with 10 times the column volume of PBS buffer. After centrifuging the serum of patients with Sjögren's syndrome at high speed, add 1 / 10 volume of 10×PBS buffer solution to mix, adjust the pH and ion concentration, and slowly add to the chromatography column. Wash with more than 10 times the column volume of PBS until no protein is detected in the effluent. Add 6 times column volume of 0.1 M glycine (pH 3.0) to elute, collect the flow-through, and neutralize with 1 / 10 volume of 1M Tris-HCl (pH 9.0). Dialyze the obtained antibody against PBS, add 50% glycerol after quantifying the antibody concentration, aliquot and store at -20°C.

Embodiment 2

[0022] Example 2 Phage display peptide library screening for SS-specific antibody-binding peptides

[0023] Ph.D.-12 peptide library kit was purchased from NEB Company, with a library capacity of 2.7×10 9 , with a titer of 1.5×10 13 / μl.. Escherichia coli ER2537 is the host strain of the peptide library. For the screening process, refer to the instruction manual of the phage display peptide library. Each round of screening was coated with total IgG in SS patient serum (10 μg per well), and 1×10 11 Phage. The degree of phage enrichment was calculated by "input / output ratio". After three rounds of screening, positive clones were selected for sequencing. The specific binding of positive phage clones to total IgG antibodies was determined by ELISA. Total IgG antibody (10 μg per well) was coated on a 96-well plate (Nunc Company) overnight at 4 °C. Pour off the unbound antibody and block with 3% BSA at 37°C for 1 hour. Put 1×10 in each hole 9 Phage were incubated at 37°C f...

Embodiment 3

[0024] Example 3 SSP and SS patient serum IgG binding detection

[0025] The synthetic peptide SSP (0.5 μg per well) was coated on a 96-well plate (Nunc Company) overnight at 4 °C. Unbound peptides were poured off and blocked with 3% BSA at 37°C for 1 hour. Serum samples from SS patients or healthy individuals were diluted 1:400, added to different wells, and incubated at 37°C for 1 hour. Wash the plate with washing solution (PBS-0.05% Tween 20) 5 times for a total of 3 minutes. 1:1000 dilution of HRP-labeled goat anti-human IgG was incubated at 37°C for 1 hour. The washing method was the same as above, and the bound anti-M13 monoclonal antibody was detected with tetramethylbenzidine (TMB, Sigma) as the substrate, and the light absorption was detected at 450 nm.

[0026] see results figure 2 , the level of SSP antibody in the serum of SS patients was significantly higher than that of healthy people. One-way ANOVA method was used for statistical analysis, and the results s...

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Abstract

The invention discloses an epitope for sicca syndrome and application thereof, and belongs to the technical field of immunological diagnosis. The epitope is a polypeptide, and an amino acid sequence of the epitope is shown as SEQ ID NO: 1. The epitope is the polypeptide, and enzyme-linked immunosorbent assay (ELISA) detection and the reaction condition of the blood serum of patients indicate that the epitope can be combined with immunoglobulin G (IgG) in the blood serum of the patients specifically and is not reacted with healthy blood. The epitope polypeptide can be used for preparing medicines for diagnosing sicca syndrome.

Description

technical field [0001] The invention belongs to the technical field of immunological diagnosis, and in particular relates to an antigenic epitope of Sjogren's syndrome and its application. Background technique [0002] Sjogren's syndrome (SS) is a systemic autoimmune disease characterized by invasion of exocrine glands such as lacrimal glands and salivary glands. The most common clinical manifestations of the disease are dry eyes and dry mouth, and can involve the lungs, kidneys, lymphatic system, blood vessels and liver and other organs. The disease has no obvious difference in regional distribution, and can occur at any age, especially postmenopausal women, with a male to female ratio of 1:9. The prevalence rate in my country is 0.77%, and the prevalence rate in the elderly population is as high as 4%. There are nearly 10 million patients in the country. Sjogren's syndrome can be divided into two types: primary Sjogren's syndrome and secondary Sjogren's syndrome. When t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/11G01N33/68
Inventor 杨光栗占国高亚萍柳川穆荣董洁刘玉
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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