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Citrulline modified peptide antigen combination and application thereof

A technology of citrulline and peptide antigen, applied in the field of biomedicine

Active Publication Date: 2018-12-07
BEIJING PROTEOME RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the diagnostic specificity of these negative rheumatoid arthritis is only 90%

Method used

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  • Citrulline modified peptide antigen combination and application thereof
  • Citrulline modified peptide antigen combination and application thereof
  • Citrulline modified peptide antigen combination and application thereof

Examples

Experimental program
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Embodiment 1

[0108] Synthesis of embodiment 1 citrulline modified peptide

[0109] 1. The sequence of the citrulline modified peptide is as follows:

[0110] GSGMSNP-(Cit)-SLEEEKY (SEQ ID NO: 1)

[0111] RLAADDF-(Cit)-LKYENEV (SEQ ID NO: 2)

[0112] NLNGGLL-(Cit)-GIEILNQ (SEQ ID NO: 3)

[0113] 2. Synthesis sequence: from the C-terminal to the N-terminal of the sequence, the steps are as follows:

[0114] 1) Weigh 0.3g of resin into a glass reactor, add DCM (dichloromethane) to swell for 30 minutes;

[0115] 2) Take out the DCM, add 0.6g of the first amino acid in the amino acid sequence, add 0.6g of DIEA (diisopropylethylamine), DMF (dimethylformamide), DCM, react by bubbling nitrogen for 60 minutes, and take out The reaction solution was washed three times by adding DMF and MEOH;

[0116] 3) Add 0.6 g of the second amino acid in the amino acid sequence to the reactor, add HBTH (1-hydroxyl, benzo, tricloxazole tetramethylhexafluorophosphate) and DIEA, react with nitrogen gas bubbling...

Embodiment 2

[0124] Example 2 Application of citrulline-modified peptides based on microsphere detection technology in the detection of patients with CCP-negative rheumatoid arthritis 1. Experimental materials and reagents

[0125] 1) Activation buffer: 0.1M NaH 2 PO 4 (Sigma-Aldrich, St. Louis, MO, USA), pH 6.2;

[0126]2) EDC solution: 50mg / mL EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride, Thermo Fisher Scientific, IL, USA);

[0127] 3) Sulfo-NHS solution: 50 mg / ml sulfo-NHS (Thermo Fisher Scientific, IL, USA);

[0128] 4) Coupling buffer: 50mM MES, pH5.0 (Sigma-Aldrich, St.Louis, MO, USA);

[0129] 5) Phosphate buffered saline PBS: 2mM KH 2 PO 4 , 10mM Na 2 HPO 4 , 137mM NaCl, 2.7mM KCl, pH7.4 (Sigma-Aldrich, St.Louis, MO, USA);

[0130] 6) Detection / cleaning buffer: 0.05% (v / v) Tween 20 dissolved in 0.1MPBS buffer;

[0131] 7) Blocking buffer: PBS-TBN, 1% BSA dissolved in 0.1MPBS buffer;

[0132] 8) Detection antibody: Alexa555-labeled anti-human IgG or anti...

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Abstract

The invention provides a citrulline modified peptide antigen combination and a composition comprising the citrulline modified peptide antigen combination. The invention also provides the use of the citrulline modified peptide antigen combination or the composition comprising the citrulline modified peptide antigen combination in preparation of a diagnostic tool detecting anti-citrulline modified peptide IgG antibodies and / or IgA antibodies. The citrulline modified peptide antigen combination provided by the invention is used for detecting IgG antibodies and / or IgA antibody markers, and solvesthe problem that 30% of clinical RA (rheumatoid arthritis) patients lack clear markers. The diagnostic tool provided by the invention adopts an antigen combination corresponding to the autoantibody ofa novel rheumatoid arthritis related biomarker to detect the autoantibody in human serum, has the advantages of good specificity, high sensitivity, good accuracy, wide linear range, safety, reliability and easy operation, and is especially suitable for screening and diagnosis of negative rheumatoid arthritis patients non-detectable by current clinical CCP2 (cyclic citrullinated peptide 2).

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to anti-citrulline-modified peptide IgG antibody and / or IgA antibody serological detection technology, in particular to citrulline-modified peptide antigen, diagnostic tool or its preparation method and CCP-negative rheumatoid Application of autoantibody detection in arthritis patients. Background technique [0002] Rheumatoid arthritis (RA, Rheumatoid Arthritis) is one of the most common systemic autoimmune diseases, which affects about 0.5-1% of the population worldwide. RA is a systemic autoimmune disease. Patients are often accompanied by chronic joint inflammation and suffer from a chronic course of the disease, which can lead to progressive joint destruction, bone erosion, and deformity. The most important serological finding of RA in rheumatoid arthritis is the presence of autoantibodies that specifically recognize citrulline-modified proteins in patient sera. About 70%...

Claims

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Application Information

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IPC IPC(8): C07K7/08G01N33/68G01N33/543
CPCC07K7/08G01N33/54313G01N33/54326G01N33/6893G01N2800/102
Inventor 于晓波王红叶
Owner BEIJING PROTEOME RES CENT
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