Biomarker and uses thereof

A technology of use and tenascin, applied in the field of biomarkers and their uses, can solve the problems of no indication of tenascin-C, regulation of the pro-inflammatory activity of tenascin-C, no indication, etc.

Inactive Publication Date: 2016-11-09
OXFORD UNIV INNOVATION LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] However, previous studies did not suggest that tenascin-C might be citrullinated, nor did it suggest that such citrullinated substances might regulate the pro-inflammatory activity of tenascin-C, or that citrullinated tenascin-C autoantigen

Method used

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  • Biomarker and uses thereof
  • Biomarker and uses thereof
  • Biomarker and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0192] Embodiment 1 general method

[0193] Reagent

[0194] Zymosan, methylated BSA and complete Freund's adjuvant, anti-FLAG M2 antibody (mouse monoclonal antibody), blasticidin, isotype control antibody (mouse IgG2a, IgG1) were obtained from Sigma-Aldrich (Dorset , UK). Hypnorm was obtained from VetaPharma Ltd (Leeds, UK). Limulus amaebocyte lysate assay was obtained from Associates of Cape Cod (Liverpool, UK). Wild-type human embryonic kidney (HEK293-EBNA) cells were obtained from Invitrogen (Groningen, Netherlands). M-CSF and murine IL-1β were obtained from PeproTech (Neuilly-Sur-Seine, France). DMEM, RPMI 1640, fetal bovine serum (FBS), penicillin / streptomycin, antibiotic-antimycotic solution PSA and β-mercaptoethanol were obtained from PAA Laboratories (Yeovil, UK). Stable expression of human TLR2 and TLR4 / CD14 / MD-2, polymyxin B, msbB LPS and function-blocked TLR2 (clone: ​​TL2.1 isotype: mouse IgG2a) and TLR4 antibody (clone: ​​HTA125 isotype: mouse IgG2a ) HEK29...

Embodiment 2

[0209] Synthesis of embodiment 2 recombinant protein

[0210] Proteins corresponding to each domain of tenascin-C (TA, EGF-L, various TNIII repeat regions and FBG) were synthesized and purified. Synthetic recombinant proteins such as Figure 9 shown.

[0211] Reagent

[0212] Pfu Turbo polymerase was obtained from Stratagene (Amsterdam, Netherlands). Easy mix 50 PCR tubes were obtained from Molecular Bioproducts (Lutterworth, UK). RNeasy kit and Ni 2+ - NTA-agarose columns were obtained from Qiagen (Crawley, UK). The pCR Blunt vector, pCEP4 plasmid vector, human embryonic kidney (HEK293-EBNA) nuclei and 4-12% Bis-Tris gradient gels were obtained from Invitrogen (Groningen, Netherlands). The pET32b vector and BL21(DE3) Rosetta cells were obtained from Novagen (Kent, UK). HiTrap Q column, HiTrap S column, Sephacryl S500HR column and Heparin Sepharose column were obtained from Amersham (Buckinghamshire, UK).

[0213] Restriction enzymes were obtained from New England BioL...

Embodiment 3

[0238] Embodiment 3 animal model

[0239] Zymosan-induced arthritis

[0240] Zymosan-induced arthritis (ZIA) was induced by injection of zymosan (Saccharomyces cerevisiae) in tenascin-C deficient and wild-type mice as described in Keystone (1977). Zymosan was prepared by dissolving 15 mg of zymosan in 1 ml of sterile PBS. The solution was boiled twice and sonicated. Mice were anesthetized by intraperitoneal injection of 150 μl of Hypnorm diluted 1:10 in sterile water followed by injection of zymosan (10 μl) into the right footpad (d=0).

[0241] Control mice were injected with only 10 μl of PBS or not. For macroscopic evaluation of arthritis, the thickness of each hind paw was measured daily using micrometer calipers (Kroeplin, Schluchlem, Germany), and the diameter was expressed as the average value of each inflamed hind paw per mouse.

[0242] After the experiment was completed (day=4), the mice were sacrificed and the hind paws were fixed with 10% (v / v) formalin, decalc...

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Abstract

The invention relates to a method of determining the inflammatory disorder status of a subject comprising detecting the presence or absence, or the level, of (i) citrullinated tenascin-C and/or one or more fragments of citrullinated tenascin-C; and/or (ii) autoantibodies with specificity for citrullinated tenascin-C and/or one or more fragments of citrullinated tenascin-C, in a sample from said subject.

Description

technical field [0001] The present invention relates to citrulline metastasin-C and its activity in chronic inflammation. In particular, the present invention relates to the use of citrulline tenascin-C and / or autoantibodies specific for citrulline tenascin-C as biomarkers for inflammatory disorders, such as rheumatoid arthritis. Background technique [0002] Inflammation is a complex biological response of tissues to harmful stimuli, such as pathogens, tissue damage, or irritants. It is a protective attempt by the tissue to remove damaging stimuli and initiate the healing process of the tissue. Abnormalities associated with inflammation comprise a large group of unrelated disorders that underlie a variety of human diseases (inflammatory diseases). Examples of diseases involving inflammation include, but are not limited to: asthma, autoimmune disease, glomerulonephritis, allergy (hypersensitivity), inflammatory bowel disease, reperfusion injury, rheumatoid arthritis, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6893G01N2440/18G01N2800/102G01N2800/52G01N33/564G01N2333/705
Inventor 金·苏珊妮·米德伍德帕特里克·约翰·维纳布尔斯
Owner OXFORD UNIV INNOVATION LTD
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