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Secondary sicca syndrome epitope 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: 2012-12-12
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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  • Secondary sicca syndrome epitope and application thereof
  • Secondary sicca syndrome epitope and application thereof
  • Secondary sicca syndrome epitope and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 sSS 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 high-speed centrifugation of the serum of patients with secondary Sjögren's syndrome, add 1 / 10 volume of 10×PBS buffer 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 sSS-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 kit. Each round of screening was coated with total IgG in sSS 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 ...

Embodiment 3

[0024] Example 3 sSSP and sSS patient serum IgG binding detection

[0025] The synthetic peptide sSSP (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 sSS 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 sSSP antibody level in the serum of sSS patients was significantly higher than that of RA, SLE, pSS patients and healthy people. Statistical analysis was performed using One...

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Abstract

The invention discloses a secondary sicca syndrome epitope and an application thereof and belongs to the technical field of immunological diagnoses. The epitope is a polypeptide, and the amino acid sequence of the epitope is shown as SEQ ID NO: 1 in the sequence table. By means of an enzyme-linked immunosorbent assay (ELISA), reaction conditions of the epitope polypeptide and patient serums are detected; and according to the detection, the epitope polypeptide can be specifically combined with intravenous gamma globulin (IgG) in the patient serums instead of reacting with serums of healthy persons. The epitope polypeptide can be used for preparing drugs for diagnosing secondary sicca syndromes.

Description

technical field [0001] The invention belongs to the technical field of immunological diagnosis, and in particular relates to a secondary Sjögren's syndrome antigen epitope and application thereof. Background technique [0002] Sjögren's syndrome is a systemic autoimmune disease mainly involving exocrine glands, clinically characterized by dry mouth and eyes, and parotid gland enlargement. Sjogren's syndrome can occur alone or secondary to other autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), etc. It has been reported that secondary Sjögren's syndrome (sSS) is different from primary Sjögren's syndrome (pSS), both have different clinical, serological and genetic features, but so far, the occurrence of secondary Sjogren's syndrome The mechanism is still unknown. [0003] Because sSS patients often have organ involvement during the course of the disease, such as diffuse interstitial lung disease, renal tubular acidosis, myositis, etc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/12G01N33/68G01N33/564G01N33/543
Inventor 杨光栗占国高亚萍李玉慧柳川穆荣董洁刘玉
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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