EP3436828A1 - Séquences de marqueurs pour la polyarthrite rhumatoïde - Google Patents

Séquences de marqueurs pour la polyarthrite rhumatoïde

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Publication number
EP3436828A1
EP3436828A1 EP17719812.4A EP17719812A EP3436828A1 EP 3436828 A1 EP3436828 A1 EP 3436828A1 EP 17719812 A EP17719812 A EP 17719812A EP 3436828 A1 EP3436828 A1 EP 3436828A1
Authority
EP
European Patent Office
Prior art keywords
seq
sequences
marker
rheumatoid arthritis
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP17719812.4A
Other languages
German (de)
English (en)
Inventor
Angelika LÜKING
Petra Budde
Peter Schulz-Knappe
Dieter ZUCHT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oncimmune Germany GmbH
Original Assignee
Protagen GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Protagen GmbH filed Critical Protagen GmbH
Publication of EP3436828A1 publication Critical patent/EP3436828A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

  • the present invention relates to a novel method for the identification of marker sequences for rheumatoid arthritis, the novel marker sequences found by means of the method and their diagnostic use. Furthermore, the present invention relates to a novel method for the identification of marker sequences for rheumatoid arthritis, the novel marker sequences found by means of the method and their diagnostic use. Furthermore, the present invention relates to a novel method for the identification of marker sequences for rheumatoid arthritis, the novel marker sequences found by means of the method and their diagnostic use. Furthermore, the
  • Marker sequences for rheumatoid arthritis in particular a protein biochip or beads and their use.
  • RA Rheumatoid arthritis
  • Diagnosis can progress the joint destruction.
  • markers for the early detection of RA or prognosis and therapy control are enormous important, especially in those patients of rheumatoid arthritis (RA), who are already in drug treatment.
  • the current classification criteria jointly published by the American College for Rheumatology (ACR) and the European League against Rheumatism (EULAR) in 2010 (Aletaha, Neogi et al., 2010), aim to identify autoantibodies (AAB) against citrullinated antigens (AAB).
  • ACR American College for Rheumatology
  • EULAR European League against Rheumatism
  • ACPA autoantibodies are present in about 75% of patients
  • the RA is considered a disease in the predominantly
  • Autoantibodies are produced against citrullinated peptides. Autoantibodies to post-translationally unmodified proteins or peptides have also been described in a few papers (Hueber, Tomooka et al., 2009, Somers, Geusens et al., 2011), but have not been systematically reviewed for new ones
  • EULAR European League against Rheumatism
  • Protein biochips are gaining an increasing industrial
  • Protein biochips require the necessary proteins to be available. In particular,
  • GATEWAY recombinational cloning application to the cloning of large numbers of open reading frames or ORFeems. Methods Enzymol, 328, 575-592).
  • ORFeems open reading frames
  • the cDNA of a particular tissue is transformed into a bacterial or eukaryotic expression vector, e.g. Yeast, cloned.
  • the vectors used for the expression are generally characterized by the fact that they carry inducible promoters, with which the timing of protein expression can be controlled.
  • expression vectors have sequences for so-called
  • Affinity epitopes or proteins on the one hand, the specific detection of the recombinant fusion proteins by means of an anti-affinity epitope
  • Antibody on the other hand becomes the specific one
  • IMAC affinity chromatography
  • expression libraries could be expressed and purified at high throughput (Braun P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E. and LaBaer, J. Biol.
  • Such protein biochips based on cDNA expression libraries are in particular the subject of WO 99/57311 and WO 99/57312.
  • RA Arthritis
  • the arrays are incubated with the serum samples and conjugated to anti-human IgG via Alexa Fluor 647
  • Auger et al. does not disclose the diagnostic use of the identified antigens.
  • EP 1 731 608 A1 discloses a method to identify "RA susceptible genes" by gene mapping using microsatellite markers and PCR technique
  • the genes were TNXB, NOTCH4 (chromosomes 6), RAB6A, MPRL48, FLJ11848, UCP2 and UCP3 (chromosome 11) found in human genomic DNA
  • EP 1 731 608 A1 claims a marker gene for an RA assay consisting of a partial DNA sequence of one of the marker genes found and comprising at least one SNP in the human genomic DNA of RA comprising recovering partial DNA sequences corresponding to one of the marker genes from a subject to be examined, determining the nucleotide sequence of the partial DNA sequence and comparing the nucleotide sequence with the corresponding nucleotide sequence derived from a
  • test kit for RA comprising one of the marker genes or one of them
  • WO 2009/138408 A2 claims a diagnostic method in which an autoantigen marker comprising the catalytic domain of BRAF or an antibody fragment thereof, for
  • Detection of RA is used and optionally also anti-PAD4 antibodies are detected in the biological sample of the subject to be examined.
  • WO 2009/138408 A2 discloses a detection kit for the detection of anti-BRAF Autoantibodies, an array of autoantigen markers comprising BRAF and PAD4 for the diagnosis of RA and the use of an autoantigen marker comprising BRAF for the diagnosis of RA, preferably in patients who are CCP negative (page 3, paragraph 1).
  • WO 2007/039280 AI claims a method for
  • Bone, cartilage or synovial membrane can be used.
  • WO 2007/039280 A1 further claims the use of a panel comprising anti-CCP and ANA for the diagnosis of RA and a test kit. Nicoaise et al. (2008) Arthritis Research Therapy, Biomed
  • Antibodies for the diagnosis of RA in CCP-negative patients and use for monitoring during therapy with infliximab are groups of patients with RA and CCP and with RA and without CCP, patients with other rheumatic Diseases (Psoriatic Rheumatism, Primary Sjögren Syndrome, Ankylosis Spondylitis) and healthy controls
  • US 2007/0254300 AI uses a yeast two-hybrid system to identify anti-inflammatory compounds ([0504] to [0506]) and claims a protein complex wherein the first protein is PAK, a fragment thereof, or a fusion protein containing the same, and the second protein ERK3, PRKAR1A, KRT23 (209), PN7098, AL117237, PCNT2, PROX1, HOOK1, IGHG1, GOLGA2, KIAA0555, LRPPRC or a fragment thereof
  • Microarray which includes this protein complex and a
  • Marker sequences for rheumatoid arthritis in particular a protein biochip and its use.
  • Marker sequences for diagnosis of RA methods for diagnosing RA using these marker sequences, methods for stratifying, an array of marker sequences, an assay / protein biochip, the use of the assembly,
  • Diagnostics comprising the marker sequences, a target for treatment and therapy, and the use of the marker sequences to perform apheresis.
  • the RA-specific expression clones were obtained in DE 10 2007 041 656 AI in that 10 or more patient samples were individually screened against a cDNA expression library and identified by comparison with 10 or more healthy samples.
  • Fig. 1 the differential
  • the object of the present invention is therefore to find better marker sequences for rheumatoid arthritis and their diagnostic use.
  • RA rheumatoid arthritis
  • the invention relates to a method for the identification of marker sequences for rheumatoid arthritis (RA), comprising the steps: a) serum samples from RA patients are contacted with more than 5,000 antigens coupled to (Luminex) beads, the binding of the individual antigens Serum RA proteins were measured by immunofluorescence assay and the median fluorescence intensity (MFI) determined for each individual antigen;
  • MFI median fluorescence intensity
  • markers are selected from the sequences SEQ ID o. 1 to 84, partial sequences or fragments thereof,
  • planar substrates are used, to the marker sequences or sequences to be examined
  • Sequences immobilized on a solid, flat support An alternative arrangement or panel of marker sequences or sequences to be examined is possible on beads, which therefore differ from conventional microarrays, inter alia with regard to their sensitivity and specificity.
  • bead arrays are either impregnated with beads at different concentrations
  • Fluorescent dye or e.g. created by barcode technology.
  • the beads are addressable and can be used to detect specific binding events occurring on their own
  • Bead technology is based on microscopically small spherical beads or platelets called microspheres or beads. These beads can be used analogously to ELISA and Western Blot as
  • Solid phase for biochemical detection reactions serve.
  • bead types which differ for example in their fluorescent color and each of which carries its own specific detection reagent on the surface. In this way, a corresponding number of different detection reactions can be carried out simultaneously in a very small sample volume.
  • Bead arrays are the specific interaction of two defined biochemical compounds detectable. Compared to conventional microarrays
  • marker sequences for RA can be identified which differ in their sensitivity and
  • the invention also relates to a marker sequence for
  • Rheumatoid arthritis obtainable by a method according to the invention and wherein the marker sequence is selected from the group of the sequences SEQ ID o. 1 to 84, partial sequences or fragments thereof, homologues of the sequences SEQ ID NO. 1 to 84 with at least 90% homology.
  • the invention also provides the use of one or more marker sequence (s) according to the invention for the diagnosis of rheumatoid arthritis.
  • One embodiment relates to the use according to the invention, wherein the marker sequence (s) on or from one to
  • One embodiment relates to the use according to the invention, characterized in that 2 or 3, preferably 4 or 5, more preferably 6, 7 or 8 or more different
  • Marker sequences for example 10 to 20 or 30 or more different marker sequences on or from one to
  • One embodiment relates to the use according to the invention, characterized in that the marker sequence (s) is / are applied to a solid support, the solid support being selected from filters, membranes, wafers, for example Silicon wafers, glass, metal, plastic, chips,
  • mass spectrometric targets for example magnetic, coated or labeled beads, such as
  • the invention also provides a method for the diagnosis of rheumatoid arthritis, wherein a. ) at least one marker sequence according to the invention is applied to a solid support, preferably to a bead, and b. ) is brought into contact with body fluid or tissue extract of a patient and
  • the detection of such an interaction can, for example, by a probe, in particular by an antibody
  • the invention also provides a method for
  • Marker sequence is used to examine a sample of the patient.
  • One embodiment relates to a method according to the invention for the diagnosis of rheumatoid arthritis, wherein the
  • the invention also relates to an arrangement or panel comprising or consisting of one or more
  • the subject of the invention is also an assay or
  • Protein array comprising an arrangement or panel according to the invention.
  • the invention is also the use of a
  • Identification and / or characterization of a substance for rheumatoid arthritis containing means for detecting a binding success, characterized in that an assembly / panel or an assay or protein array is contacted with a.) At least one substance to be examined and b.) Demonstrates a binding success becomes.
  • the invention also provides a diagnostic agent for
  • Diagnosis of rheumatoid arthritis containing at least one marker sequence according to the invention and optionally further excipients and additives.
  • the invention also provides a target for the treatment or therapy of rheumatoid arthritis, wherein the target is selected from the marker sequences according to the invention.
  • the invention also provides the use of at least one marker sequence according to the invention for identifying a subgroup of patients within the group of
  • Marker sequences for the diagnosis of rheumatoid arthritis wherein at least one marker sequence selected from the group of marker sequences SEQ ID o. 1 to 84 and / or genomic sequences containing one of the sequences SEQ ID NO. 1 to 42 and / or a by the sequences SEQ ID No. 43 to 84 coded protein, partial sequences or fragments thereof, homologues of the sequences SEQ ID NO. 1 to 84 with at least 90% homology to or from a patient to be examined.
  • Another embodiment of the invention relates to
  • marker sequence (s) for the diagnosis of rheumatoid arthritis, characterized in that the determination is carried out by means of in-vitro diagnosis.
  • Marker sequences for rheumatoid arthritis can be identified.
  • Beads (beads, globules, originally also called latex particles) denote so-called microspheres or
  • Microparticles used as carriers for biomolecules in tests and assays are required, which are produced by special chemical processes. These methods are known to the person skilled in the art. Beads for different applications are also commercially available (eg Fa. Progen Biotechnik GmbH) . Beads can be made of different materials, for example glass, polystyrene, PMMA and
  • Beads can be labeled with different dyes or dye mixtures and provided with coatings. At yours
  • the surface of the beads may be modified such that directional coupling of the biomolecules on the bead surface, e.g. in connection with spacers, tags or special modifications is possible and whereby the
  • rheumatoid arthritis is for example after
  • Juvenile idiopathic arthritis (ICD-10: M08.-, abbr .: JIA) Older synonyms: Juvenile rheumatoid arthritis, juvenile chronic arthritis, Still's disease or more popular: childlike
  • the marker sequences according to the invention can also be combined, supplemented, fused or extended with known biomarkers for this indication.
  • the determination of the marker sequences takes place outside the human body and the determination takes place in an ex vivo / in vitro diagnosis.
  • Diagnosis in the sense of this invention means the positive detection of rheumatoid arthritis by means of
  • Marker sequences of the invention and the assignment of patients to the indication rheumatoid arthritis.
  • diagnosis includes medical diagnostics and
  • diagnosis also includes the
  • the invention relates to a method for
  • Marker sequence according to the invention is determined on a patient to be examined. Also included is the
  • therapy control also includes the classification of patients into responders and non-responders with regard to a therapy or its course of therapy.
  • stratification also: stratification
  • therapy control in the sense of this invention means that the inventive method decisions for the treatment and therapy of the
  • the term "stratification" includes in particular the
  • patient is understood to mean any subject - human or mammal - with the proviso that the subject is being examined for rheumatoid arthritis.
  • marker sequences in the sense of this invention means that the nucleic acid sequence, for example the mRNA, cDNA or the respective polypeptide or protein obtainable therefrom, are significant for rheumatoid arthritis.
  • the mRNA or cDNA or the respective polypeptide or protein obtainable therefrom may interact with substances from the body fluid or tissue extract of a rheumatoid arthritis patient (e.g., (auto) antigen (epitope) / (auto) antibody (paratope).
  • substances from the body fluid or tissue extract of a rheumatoid arthritis patient e.g., (auto) antigen (epitope) / (auto) antibody (paratope).
  • Marker sequence selected from the group of marker sequences SEQ ID o. 1 to 84 and / or genomic sequences, the one of the sequences SEQ ID o. 1 to 42 and / or a by the sequences SEQ ID No. 43 to 84 coded protein,
  • a binding in particular a binding substance to at least one of the marker sequences according to the invention or in the case of a cDNA hybridization with a suitable
  • Hybridization conditions are: hybridization in 4 x SSC at 65 ° C (alternatively in 50% formamide and 4X SSC at 42 ° C), followed by several washes in 0.1 x SSC at 65 ° C for a total of about one hour.
  • An example of less stringent hybridization conditions is hybridization in 4 x SSC at 37 ° C, followed by several washes in 1 x SSC
  • such substances are constituents of a body fluid, in particular blood, whole blood, blood plasma, blood serum, patient serum, urine, cerebrospinal fluid, synovial fluid or a tissue extract of the patient.
  • the marker sequences according to the invention may be present in a significantly higher or lower expression rate or concentration in the subjects are present, compared to the expression rate or concentration of the subject
  • Marker sequence in a healthy or a subject without RA is an indication of rheumatoid arthritis and the diagnosis of RA.
  • Marker sequences according to the invention can be determined, for example, by means of proteomics or nucleic acid blots.
  • the protein is preferred for a protein
  • the marker sequences according to the invention recognize autoantibodies which are specific for RA or which in the formation and the
  • Progression of RA disease can be formed and / or increased or decreased.
  • Two or more marker sequences according to the invention can be used to detect autoantibody profiles or changes in
  • SEQ ID no. 1 to 84 are the subject of Table 1 and can by the respective
  • the invention also relates to the full-length sequences of the marker sequences according to the invention and the marker sequences as defined in the tables about the known database entries as well as the attached sequence listing
  • marker sequences in particular of the nucleic acid sequences SEQ ID No. 1 to 42 and the protein sequences SEQ ID No. 43 to 84.
  • Preferred marker sequences having P values less than or equal to 0.006, preferably less than or equal to 0.001 or less than or equal to 0.0001, more preferably less than or equal to 0.00001 (see Tables 2 and 3).
  • SEQ ID No. 4 and 46 DCTN1 with reactivity in all patient cohorts.
  • SEQ ID no. 5 and 47 GPTG
  • SEQ ID NO. 6 and 48 HNRNPA1
  • SEQ ID No. 7 and 49 IFG3
  • an arrangement or panel containing at least one sequence selected from the group SEQ ID no. 4 and 46 (DCTN1), SEQ ID No. 5 and 47 (GNPTG), SEQ ID No. 6 and 48 (HNRNPA1) and SEQ ID No. 7 and 49 (ITFG3) and optionally further marker sequences according to the invention.
  • homologs of the marker sequences according to the invention are included.
  • Homologues can be protein or
  • nucleic acid sequences are those sequences which are 50 to 100 nucleotides or amino acids, preferably 70-120
  • Nucleotides or amino acids particularly preferably 100 to 200 nucleotides or amino acids of one of the marker sequences SEQ ID No. 1 to 84.
  • the marker sequences also include such
  • nucleotide sequence for example the cDNA sequence and the corresponding amino acid sequence, such as
  • Marker sequence may be represented in different amounts in one or more areas on a solid support, for example a bead. This allows a variation of the sensitivity.
  • the regions may each comprise a total of marker sequences, i. a sufficient number
  • marker sequences in particular 2 to 5 or 10 or more marker sequences and, if appropriate, further nucleic acids and / or proteins, in particular biomarkers. However, at least 96 to 25,000 (numerically) or more of different or identical marker sequences and more are preferred
  • Nucleic acids and / or proteins in particular biomarkers. Further preferred are more than 2,500, particularly preferred
  • arrangement or “panel” synonymously means “array” and insofar as this "array” is used to identify substances to be bound to marker sequences, This is to be understood as meaning an “assay” or a diagnostic device
  • arrangement is designed in such a way that that on the device
  • marker sequences in the form of a grid on a solid support. Furthermore, such arrangements are preferred which allow a high density array of marker sequences and spotting the marker sequences. Such high density spotted assemblies are
  • the term "assay" or diagnostic device also includes such embodiments of a device, such as ELISA (e.g., individual wells
  • Examples are diagnostic ELISA kits from the company Phadia or multiplex ELISA kits
  • Marker sequences according to the invention or combinations of marker sequences are robot-supported on membranes
  • Example “Euroline” from Euroimmun AG Western Blot (example “Euroline-WB” from Euroimmun AG), immunochromatographic methods (eg lateral flow immunoassays, marker sequences or combinations of marker sequences are applied to test strips (membranes, US Pat
  • One or more marker sequences may be present several times in the totality of all marker sequences and be present in different amounts relative to one spot. Furthermore, the marker sequences can be standardized on the solid support (e.g., by serial dilution series of, e.g., human globulins as internal calibrators to
  • the invention therefore relates to an assay or protein biochip or one or more beads (bead-based assay) consisting of an array or panel containing marker sequences according to the invention.
  • the marker sequences are present as clones.
  • Such clones can be obtained, for example, by means of a cDNA expression library according to the invention (Büssow et al., 1998 (supra)).
  • such expression libraries become
  • These expression vectors preferably contain inducible promoters. The induction of
  • Expression can e.g. by means of an inductor, such as IPTG.
  • IPTG inductor
  • Suitable expression vectors are described in Terpe et al. (Terpe T Appl Microbiol Biotechnol 2003 Jan; 60 (5): 523-33).
  • Expression libraries are known to the person skilled in the art, these can be prepared according to standard works, such as Sambrook et al., Molecular Cloning, Laboratory Laboratory, 2nd edition (1989), CSH press, Cold Spring Harbor, New York Further preferred are such expression libraries
  • tissue specific e.g., human tissue, especially human organs.
  • expression libraries are also included according to the invention, which can be obtained by exon trapping. Instead of expression library can be spoken synonymously from an expression bank.
  • protein biochips or beads or corresponding expression libraries which have no redundancy (so-called: UnicloneO library) and according to the teachings of WO 99/57311 and WO 99/57312, for example
  • Libraries have a high content of non-defective, fully expressed proteins of a cDNA expression library.
  • the clones may not be such as transformed bacteria, recombinant phage or transformed cells of mammals, insects, fungi, yeasts or plants.
  • the clones are fixed on a solid support, spotted or immobilized. Therefore, the invention relates to an arrangement, wherein the
  • Marker sequences are present as clones.
  • marker sequences may be in the form of a fusion protein in the particular form
  • the tag may be one such as c-myc, His-tag, Arg-tag, FLAG, alkaline phosphatase, V5 tag, T7 tag or Strep tag, HAT tag, NusA, S-tag, SBP tag , Thioredoxin, DsbA, a fusion protein, preferably a cellulose-binding one
  • Protein Protein, calmodulin-binding protein, glutathione S-transferase or lacZ.
  • a marker sequence can also be made up of several individual ones
  • marker sequences This may involve cloning individual fragments into a large common fragment and expressing this combined fragment.
  • solid support includes embodiments such as a filter, a membrane, a magnetic or fluorophore-labeled bead
  • Silicon wafer, glass, metal, plastic, a chip, a mass spectrometric target or a matrix Silicon wafer, glass, metal, plastic, a chip, a mass spectrometric target or a matrix.
  • a filter and beads are preferred according to the invention.
  • PVDF polyvinyl styrene
  • nitrocellulose polymethyl methacrylate
  • nylon polymethyl methacrylate
  • filters eg Immobilon P Millipore, Protran Whatman, Hybond N + Amersham.
  • this corresponds to a grid having the order of a microtiter plate (8-12 wells strips, 96 wells, 384 wells or more), a silicon wafer, a chip, a mass spectrometric target or a matrix.
  • beads are used as beads.
  • bead-based multiplex assays are used.
  • the analysis and evaluation of the bead-based assays can be carried out, for example, with a Luminex analysis system, which is carried out on the method of flow cytometry using two different lasers.
  • CVs coefficients of variation
  • Luminex ie bead-based protein arrays
  • the UNIarray concept is not bound to Luminex, but can also be used on other platforms such as Randox, VBC-Genomics etc.
  • the high measurement accuracy and the low VKs of the individual measurements allow the use of better and new statistical
  • the invention relates to an assay or protein biochip for identifying and
  • Characterizing a substance for rheumatoid arthritis characterized in that an arrangement or assay according to the invention is brought into contact with a.) At least one substance to be investigated, and b.) A binding success
  • the substance to be tested may be any native or non-native biomolecule
  • Substance library After the substance to be examined contacts a marker sequence, the binding success is evaluated, which is carried out, for example, using commercially available image analysis software (GenePix Pro (Axon Laboratories), Aida (Raytest), ScanArray (Packard Bioscience).
  • protein to marker sequence e.g., protein to marker sequence
  • Antigen / antibody or corresponding "means for detecting the binding success" can, for example, by means of
  • Fluorescence labeling, biotinization, radioisotope labeling or colloidal gold or latex particle Marking done in the usual way. Detection of bound antibodies takes place with the help of secondary antibodies
  • reporter enzymes such as alkaline
  • a readout is e.g. by means of a microarray laser scanner, a CCD camera or visually.
  • Protein biochips each performed from a cDNA expression library of a patient and a healthy subject and the differential clones are detected by fluorescence labeling and bioinformatorisch evaluated.
  • the 6,000 antigens were recombinantly expressed in E. coli and purified.
  • 5 cDNA banks (oligo (dT) primed, His-tag) from human tissue were used (inter alia, intestine, lung, liver)
  • Vidalain PO Clingingsmith TR, Hartley JL, Esposito D, Cheo D, Moore T, Simmons B, Sequerra R, Bosak S, Doucette Tribe L, Le Peuch C, Vandenhaute J, Cusick ME, Albala JS, Hill DE, Vidal M : Human ORFeome version 1.1: a platform for reverse proteomics. Genome Res 2004, 14: 2128-2135). recombinant
  • Escherichia coli is dependent on the availability of the DNA gene product. 1989, 85: 109-114). Proteins were obtained from harvested and lysed cells (Overnight Express autoxidation medium, Novagen®, 6M guanidinium HCl, 0.1M
  • Luminex Corporation conducted according to Luminex protocol, wherein for each single-coupling reaction up to 12.5 pg antigen and 8.8 x 105 MagPlexTM beads of one color region (ID)
  • Cohort C (seronegative RA cohort): 184 patients with ACPA-negative RA according to 2010 ACR / EULAR criteria (age 60.2 ⁇ 13.8 years, 62.5%% female, all therapy naive) compared with 343 healthy controls 343 (age 47.7 ⁇ 11.7years, 58.3 %% female).

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Abstract

La présente invention concerne une nouvelle méthode d'identification de séquences de marqueurs pour la polyarthrite rhumatoïde, les nouvelles séquences de marqueurs identifiées par ce procédé, et leur utilisation diagnostique. L'invention concerne en outre des dispositifs diagnostiques contenant de telles séquences de marqueurs de la polyarthrite rhumatoïde, notamment une biopuce à protéines ou des microbilles (beads) et leur utilisation.
EP17719812.4A 2016-04-02 2017-04-03 Séquences de marqueurs pour la polyarthrite rhumatoïde Withdrawn EP3436828A1 (fr)

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EP16163611 2016-04-02
EP16170028 2016-05-17
PCT/EP2017/057896 WO2017168014A1 (fr) 2016-04-02 2017-04-03 Séquences de marqueurs pour la polyarthrite rhumatoïde

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