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Polypeptide combined with immune antibody and application thereof

An antibody and in vitro detection technology, applied to the peptides combined with immune antibodies and their application fields, can solve the problems of incomparable diagnostic sensitivity of rheumatoid arthritis, no data to support BiP in predicting rheumatoid arthritis, etc., and achieve high affinity Effect

Active Publication Date: 2012-07-18
SHANGHAI RONGSHENG BIOLOGICAL PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

64% of patients with rheumatoid arthritis can produce antibodies directly against BiP, and there are reports that it is highly specific to the disease. There is currently no data to support the role of BiP in predicting rheumatoid arthritis, and these reports BiP antibodies need to wait for further confirmation through independent clinical research (Clinica ChimicaActa 2004, 350, 17-34)
It must be noted that the sensitivity of RF in the ACR criteria for the diagnosis of rheumatoid arthritis cannot be directly compared with those of other methods not included in the diagnostic criteria

Method used

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  • Polypeptide combined with immune antibody and application thereof
  • Polypeptide combined with immune antibody and application thereof
  • Polypeptide combined with immune antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Synthesis of Polypeptides

[0080]Polypeptides are synthesized by solid-phase synthesis technology using Fmoc chemical method. For the specific steps of this method, see Eur.J.Immunol.1994, 24, 3188-3193; J.Org.Chem.1972, 37, 3404-3409; Huang Weide, Chen Changqing Polypeptide Synthesis, Beijing: Science Press, 1985.

[0081] The formation method and steps of disulfide bonds can be found in the literature: Huang Weide, Chen Changqing Peptide Synthesis, Beijing: Science Press, 1985, p85; Michael W.Pennington Peptide Synthesis Protocols (Methods in Molecular Biology), Humana Press, 1994, p91-169

[0082] Through the above steps, the specific sequence of the synthetic polypeptide is:

[0083] Polypeptide 1: His-Glu-Cys-His-Glu-Phe-Arg-Phe-Cit-Gly-Cit-Ser-Arg-Ala-Ala-Cys-Glu (SEQ ID No 1), the third and the first Sixteen-position Cys forms a disulfide bond through a sulfhydryl group, and makes the polypeptide form a ring structure, and can simulate the β-turn str...

Embodiment 2

[0085] Example 2 Purification of polypeptides

[0086] First, the protecting group at the end of the protein or polypeptide is deprotected and neutralized in dimethylamide (DMF) solvent. The polypeptide is cut off from the synthetic resin with hydrogen fluoride, and the obtained crude product is passed through a reversed-phase chromatography C18 or C8 column (such as: 5 μm, 250×4.6 mm), and the mobile phase A (0.1% (v / v) trifluoroacetic acid Acetonitrile solution) and mobile phase B (0.1% (v / v) trifluoroacetic acid in water) were gradient elution solvents. Within 45 minutes, the mobile phase A accounted for the total volume of the two phases A and B changed from 0% (v / v) to 100% (v / v) to collect the target polypeptide, and the organic phase was removed by desalting chromatographic column (GE Healthcare) or rotary evaporation. The solvent and the target polypeptide can be determined by combining LC-MS or directly injecting the collected polypeptide through the molecular weight...

Embodiment 3

[0087] Example 3 The purity and molecular weight detection of polypeptide

[0088] The above synthesized polypeptide 1 and polypeptide 2 were determined by reverse phase chromatography (RP-HPLC) respectively, and the specific method is as follows:

[0089] 4.6×250mm 5μm C18 analytical column (Kromasil);

[0090] Mobile phase A is trifluoroacetic acid (trifluoroacetic, TFA) added to 100% acetonitrile (acetonitrile, ACN), so that the concentration of TFA is 0.1% (v / v);

[0091] Mobile phase B is TFA added to 100% water, so that the concentration of TFA is 0.1% (v / v);

[0092] The flow rate is 1.0ml / min;

[0093] The detection wavelength is 220nm;

[0094] Elution gradient: The ratio of mobile phase A to the total volume of A and B is 15% (v / v). After injection, linear gradient elution (Gradient Elution) is used, and mobile phase A accounts for A within 25 minutes. The ratio of the total volume of the two items of B and B was changed from 15% (v / v) to 50% (v / v), and then equi...

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Abstract

The invention discloses a polypeptide, wherein the sequence thereof contains at least one arginine side chain modified to be electric neutral or electric negative amino acid. Two non-adjacent cysteine side chain sulfydryl groups on the sequence form a disulfide bond, thus producing annular polypeptide. The polypeptide not only can be combined with rheumatoid arthritis autoimmunity antibody but also has high appetency on HLA-DR. Experiment shows that the combination specificity of the polypeptide and the rheumatoid arthritis autoimmunity antibody is more than 95% and detection sensitivity on the rheumatoid arthritis autoimmunity antibody is more than 75%. Compared with like products sold on market, the sensitivity thereof is obviously improved, thus being more beneficial to in vitro detection of rheumatoid arthritis autoimmunity antibody.

Description

technical field [0001] The present invention relates to a polypeptide that binds to immune antibodies, in particular to a polypeptide that binds to rheumatoid arthritis immune antibodies, and more specifically relates to a polypeptide that binds to rheumatoid arthritis autoimmune antibodies with at least one arginine modified of polypeptides. Background technique [0002] Clinically, autoimmune diseases mainly include systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, dermatomyositis, polymyositis, systemic (multiple) sclerosis, scleroderma, etc. These diseases were once named " Connective tissue disease", later both abroad and domestically classified them as rheumatic diseases. [0003] Rheumatoid arthritis (RA) is a relatively common systemic autoimmune disease mainly manifested by chronic polyarticular inflammation. Great pain; the incidence rate of the disease is 0.5-1% worldwide, and the incidence rate in China is about 0.36%. The age of onset of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08G01N33/68
Inventor 王绍成朱绍荣
Owner SHANGHAI RONGSHENG BIOLOGICAL PHARM CO LTD
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