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Epitope of systemic lupus erythematosus and application thereof

A lupus erythematosus and antigenic epitope technology, applied in the field of immunological diagnosis, can solve the problems of unclear pathogenesis, self-antigen detection antibody, etc.

Active Publication Date: 2012-10-03
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 of systemic lupus erythematosus and application thereof
  • Epitope of systemic lupus erythematosus and application thereof
  • Epitope of systemic lupus erythematosus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 SLE 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 the serum of patients with systemic lupus erythematosus was centrifuged at high speed, 1 / 10 volume of 10×PBS buffer solution was added to mix, and the pH and ion concentration were adjusted, then slowly added 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 SLE-specific antibody-binding peptides

[0023] Ph.D.-12 peptide library kit was purchased from NEB Company, with a library capacity of 2.7×109 and a titer of 1.5×1013 / μl. Escherichia coli ER2537 was used as the host bacteria 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 the serum of SLE patients (10 μg per well), and 1×1011 phage were injected into each well. 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 1×109 phage...

Embodiment 3

[0024] Example 3 SLEP and SLE patient serum IgG binding detection

[0025] The synthetic peptide SLEP (0.5 μg per well) was coated on a 96-well solid microtiter plate (Nunc Company) overnight at 4 °C. Unbound peptides were poured off and blocked with 3% bovine serum albumin (BSA) at 37°C for 1 hour. Serum samples from SLE 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 SLEP antibody in the serum of patients was significantly higher than that of healthy people. Statistical analysis was perfor...

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Abstract

The invention discloses an epitope of systemic lupus erythematosus and an application thereof, belonging to the technical field of immunology diagnosis. The epitope is a polypeptide of which the amino acid sequence is shown as a sequence table SEQ ID NO:1. As proved by ELISA (Enzyme-Linked Immunosorbent Assay) detection and the serum reaction condition of a patient, the epitope polypeptide can be specifically combined with IgG (Immunoglobulin G) in the serum of the patient without reacting with the serum of a healthy person. The epitope polypeptide can be used for preparing a medicament for diagnosing systemic lupus erythematosus.

Description

technical field [0001] The invention belongs to the technical field of immunological diagnosis, and in particular relates to an antigenic epitope of systemic lupus erythematosus and its application. Background technique [0002] Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies and the formation of immune complexes. The clinical manifestations are systemic symptoms and symptoms of multi-system and multi-organ involvement. The incidence rate of SLE in my country is 70.1 / 100,000, and there is an increasing trend year by year. Early diagnosis and early treatment of SLE are the key to improve the prognosis. However, the diagnosis level of autoimmune diseases in my country is relatively low, and diagnostic reagents are mainly imported, resulting in high diagnostic costs, increasing the medical costs of the country and patients, and not conducive to popularization in primary hospitals. Therefore, it is an urgent problem...

Claims

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Application Information

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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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