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Embodiment 1
[0074] Synthesis of embodiment 1 citrulline modified peptide
[0075] 1. The sequence of the citrulline modified peptide is as follows:
[0076] GSGMSNP-(Cit)-SLEEEKY (SEQ ID NO: 1)
[0077] 2. Synthesis sequence: from the C-terminal to the N-terminal of the sequence, the steps are as follows:
[0078] 1) Weigh 0.3g of resin into a glass reactor, add DCM (dichloromethane) to swell for 30 minutes;
[0079] 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;
[0080] 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 for 30 minutes, and wash off the liquid , ninhydrin detection, followed by capping with pyri...
Embodiment 2
[0088] 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
[0089] 1) Activation buffer: 0.1M NaH 2 PO 4 (Sigma-Aldrich, St. Louis, MO, USA), pH 6.2;
[0090] 2) EDC solution: 50mg / mL EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride, Thermo Fisher Scientific, IL, USA);
[0091] 3) Sulfo-NHS solution: 50 mg / ml sulfo-NHS (Thermo Fisher Scientific, IL, USA);
[0096] 8) Detection antibody: Alexa555-labeled anti-human IgG or ant...
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Abstract
The invention provides a citrulline modified peptideantigen and a composition comprising the citrulline modified peptideantigen. The invention also provides the use of the citrulline modified peptideantigen or the composition comprising the citrulline modified peptide antigen in preparation of a diagnostic tool detecting anti-citrulline modified peptide IgG antibodies and / or IgA antibodies. Thecitrulline modified peptide antigen provided by the invention is used for detecting IgG antibodies and / or IgA antibody markers, and solves the problem that 30% of clinical RA (rheumatoid arthritis) patients lack clear markers. The diagnostic tool provided by the invention adopts an antigen corresponding to the autoantibody of a 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%...
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