WO2006114661A1 - Analyse de glycane a haut rendement pour diagnostiquer et surveiller l'arthrite rhumatoide et d'autres maladies auto-immunes - Google Patents

Analyse de glycane a haut rendement pour diagnostiquer et surveiller l'arthrite rhumatoide et d'autres maladies auto-immunes Download PDF

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WO2006114661A1
WO2006114661A1 PCT/IB2005/002885 IB2005002885W WO2006114661A1 WO 2006114661 A1 WO2006114661 A1 WO 2006114661A1 IB 2005002885 W IB2005002885 W IB 2005002885W WO 2006114661 A1 WO2006114661 A1 WO 2006114661A1
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glycans
autoimmune disease
glycoproteins
glycosylation
glycosylation profiles
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PCT/IB2005/002885
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English (en)
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Raymond A. Dwek
Louise Royle
Pauline Rudd
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Dwek Raymond A
Louise Royle
Pauline Rudd
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Priority to US11/411,231 priority Critical patent/US20060269979A1/en
Publication of WO2006114661A1 publication Critical patent/WO2006114661A1/fr

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • This invention is directed to diagnostic and monitoring methods for rheumatoid arthritis and other autoimmune diseases and, in particular, to diagnostic and monitoring methods for rheumatoid arthritis (RA) and other autoimmune diseases based on detailed glycosylation analysis of glycoprotein glycans.
  • RA rheumatoid arthritis
  • RA is generally considered a systemic inflammatory disease in which an immune response by the adaptive immune system translates into an attack on the diarthrodial joints (synovium, cartilage, and bone with attendant joint destruction) and less frequently on other anatomic sites.
  • lymphocytes in RA pathogenesis. Histologically, T- cells account for a portion of the mononuclear infiltrate in the synovial sublining, see Van Boxel, J. A., and S. A. Paget. Predominantly T-cell infiltrate in rheumatoid synovial membranes. New England Journal of Medicine 293:517, 1975.
  • the Sa system a novel antigen-antibody system specific for rheumatoid arthritis
  • J Rheumatol 21:1027, 1994 BiP (Blass, S., Novel 68 kDa autoantigen detected by rheumatoid arthritis specific antibodies. Ann Rheum Dis 54:355, 1995)
  • RA33 Hassfeld, W., G. Steiner, K. Hartmuth, G. Kolarz, O. Scherak, W. Graninger, N. Thumb, and J. S. Smolen. Demonstration of a new antinuclear antibody (anti-RA33) that is highly specific for rheumatoid arthritis.
  • T-lymphocytes B-cells are frequently found in the synovial mononuclear infiltrate in RA. With discrete differences, these lymphocytes can organize into aggregates similar to those found in lymph nodes and Peyer's patches (Rooney, M., A. et.al. The immunohistologic features of synovitis, disease activity and in vitro IgM rheumatoid factor synthesis by blood mononuclear cells in rheumatoid arthritis. Journal of Rheumatology 16:459, 1989). Taken together, these findings implicate autoimmunity involving T-lymphocytes, B-lymphocytes and IgG in the pathogenesis of RA.
  • immunoglobulin G of serum or other body fluid.
  • Methods for diagnosing and monitoring diseases based on mass-spectrometric measuring of glycosylation profiles of glycans released from purified glycoproteins are also disclosed in US patent application publication "Glycan Markers for Diagnosing and Monitoring Disease" No. 2004/0147033 to Shriver et. al. published on July 29, 2004.
  • One embodiment of the invention is a method for diagnosing and monitoring an autoimmune disease comprising releasing glycans of glycoproteins from samples of a body fluid without purifying the glycoproteins, and without exposing the body fluid to hydrazinolysis; and quantitatively analyzing the glycans.
  • the method may further be used to improve therapy for an autoimmune disease by establishing optimal dosage for an existing therapeutic agent used to treat the autoimmune disease.
  • the glycosylation profile during treatment of an autoimmune disease patient is monitored to assess whether different dosages of a therapeutic agent change the glycosylation profile so that it moves closer to the glycosylation profile of a normal individual.
  • the method may further be used to screen for new therapeutic agents by generating a candidate agent to be assessed for therapeutic activity in the treatment of an autoimmune disease and determining whether the candidate agent changes the glycosylation profile in an autoimmune disease patient so that it moves closer to the glycosylation profile of a normal individual, hi this regard, combinatorial chemistry may be used to rapidly generate candidate agents for screening in the method of the present invention to determine therapeutic activity in the treatment of an autoimmune disease.
  • Another embodiment of the invention is a method of diagnosing and monitoring an autoimmune disease comprising measuring diseased glycosylation profiles and one or more control glycosylation profiles, wherein the diseased glycosylation profiles are glycosylation profiles of glycans of glycoproteins from autoimmune disease patients and the one or more control glycosylation profiles are glycosylation profiles of glycans of glycoproteins from patients without the autoimmune disease; and comparing peak ratios in the diseased glycosylation profiles and in the one or more control glycosylation profiles and selecting a ratio having a highest correlation with parameters of the autoimmune disease patients out of the peak ratios as a glycosylation marker of the autoimmune disease.
  • Yet another embodiment of the invention is a high throughput method for diagnosing and monitoring rheumatoid arthritis in a patient comprising releasing glycans of glycoproteins from a body fluid or a body tissue of the patient; and measuring a ratio between an amount of GO glycans and an amount of Gl glycans in the glycans.
  • FIGURE 1 shows sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE) and normal phase high performance liquid chromatography (NP-HPLC) profiles of glycans released from purified immunoglobulin G (IgG) of samples GBRA13 and GBRAl.
  • SDS-PAGE sodium dodecyl sulphate polyacryl amide gel electrophoresis
  • NP-HPLC normal phase high performance liquid chromatography
  • FIGURE 2 shows NP-HPLC profiles of glycans released from purified IgG of sample GBRAl 5.
  • FIGURE 3 shows NP-HPLC profiles of control and sample GBRAl 5.
  • FIGURE 4 shows a correlation between GO/tripleGl versus GO as a percentage of total purified IgG glycans for purified IgG glycans.
  • FIGURE 5 shows a correlation between GO/tripleGl from serum versus purified IgG.
  • FIGURE 6 shows a correlation between GO/tripleGl for glycans released from whole serum and GO as a percentage of total glycans released from purified IgGs.
  • FIGURE 7 shows GO/tripleGl ratios in glycans released from whole serum using polyvinyldene fluoride (PVDF) membranes (serum PVDF) and in glycans released from purified IgG heavy chain gel bands (purified IgG heavy chain gel band).
  • PVDF polyvinyldene fluoride
  • the present invention is directed to diagnostic and monitoring methods for autoimmune diseases and, in particular, to diagnostic and monitoring methods for autoimmune diseases based on detailed glycosylation analysis of glycans of glycoproteins.
  • One embodiment of the invention is a method for diagnosing and monitoring an autoimmune disease comprising releasing glycans of glycoproteins from samples of body fluid without purifying the glycoproteins and without exposing the body fluid to hydrazinolysis; and quantitatively analyzing the glycans.
  • the method of the invention can be also used for prognosticating and predicting response to specific therapies in a patient of the autoimmune disease.
  • the autoimmune disease can be, for example, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, systematic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, psoriatic arthritis, multiple sclerosis, inflammatory bowel disease, graft-vs-host disease or scleroderma.
  • the methodology of the invention can be also applied to other diseases associated with glycosylation changes, for example, to congenital disorders of glycosylation and cancers.
  • Glycans can be released from a sample of a body fluid or a body tissue, such as a sample of whole serum, blood plasma, urine, seminal fluid, seminal plasma, feces or saliva.
  • the released glycans can be N-glycans or O-glycans.
  • releasing a glycan pool of glycoproteins from a sample of a body fluid or a body tissue can be carried out without purifying the glycoproteins.
  • the released glycans are glycans of all or substantially all of the glycoproteins present in the sample of a body fluid or a body tissue rather than of one or more purified and isolated glycoproteins.
  • substantially all of the glycoproteins can mean all the glycoproteins that are recovered, yet in some embodiments substantially all of the glycoproteins can mean all the glycoproteins except those that are specifically removed.
  • Releasing glycans can be carried out without exposing a sample of a body fluid or a body tissue to hydrazinolysis. In some embodiments, releasing glycans can be carried out from a very small sample of a body fluid. In some embodiments, samples of a body fluid can be less than 100 microliters, yet preferably less than 50 microliters, yet more preferably less than 20 microliters, yet more preferably less than 10 microliters, yet most preferably less than 5 microliters.
  • releasing glycans can comprise releasing glycans from total glycoproteins of a body fluid or a body tissue in solution.
  • releasing glycans can comprise immobilizing total glycoproteins of a body fluid or a body tissue, for example, on protein binding membrane or in a gel.
  • IProtein binding membrane can be any protein binding membrane, for example, polyvinyldene fluoride (PVDF) membrane, nylon membrane or Polytetrafiuoroethylene (PTFE) membrane.
  • releasing glycans can further comprise releasing glycans from the total glycoproteins immobilized on the protein binding membrane or in the gel.
  • releasing glycans from the immobilized glycoproteins can be carried out using enzymatic release with, for example, peptide N glycosidase F.
  • releasing glycans can comprise separating the gel into a plurality of bands and selecting one or more bands from the plurality of bands from which the glycans are subsequently released (in gel band method).
  • releasing glycans from the gel can be carried out from the total gel, i.e. without separating gel into the bands.
  • releasing glycans is carried out by chemical release methods, such as / ⁇ -elimination or ammonia-based / ⁇ -elimination, which can be used for releasing JV-linked or O-linked glycans from glycoproteins in solution or from glycoproteins immobilized on protein binding membrane.
  • chemical release methods such as / ⁇ -elimination or ammonia-based / ⁇ -elimination, which can be used for releasing JV-linked or O-linked glycans from glycoproteins in solution or from glycoproteins immobilized on protein binding membrane.
  • In-gel-band This method can be used for N-glycan release from single glycopeptides in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) gel bands and is based on the method described in Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A. and Harvey, D. J. (1997) "Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high- performance liquid chromatography.” Anal-Biochem 250: 82-101, incorporated hereby by reference in its entirety.
  • Samples can be reduced and alkylated by adding 4 ⁇ l of 5X sample buffer (5X sample buffer: 0.04g Bromophenol blue, 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water), 2 ⁇ l of 0.5M dithiothreitol (DTT) and water to make up to 20 ⁇ l in total, incubated at 7O 0 C for lOmin, then alkylated by addition of 2 ⁇ l of 10OmM iodoacetamide and incubated for 30 min in the dark at room temperature.
  • 5X sample buffer 0.04g Bromophenol blue, 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water
  • DTT dithiothreitol
  • Samples can be then separated on SDS-PAGE gels after which the proteins are stained with Coomassie brilliant blue, the band of interest is excised and destained. Subsequently, the gel band can be cut into lmm pieces and frozen for 2 hours or more (this can help break down the gel matrix). This gel band can be then washed alternatively with ImI of acetonitrile then ImI of digestion buffer (2OmM NaHCO 3 pH 7), which can be repeated twice before the gel plug can be then dried. PNGase F buffer solution (30 ⁇ l of 100 U/ml) is added (this is enough for 10- 15mm 3 gel), more enzyme solution is added if larger gel bands can be used. The PNGaseF and gel pieces can be incubated overnight at 37 0 C.
  • the supernatant can be recovered along with 3 x 200 ⁇ l water washes (with sonication with gel pieces for 30 mins each) followed by an acetonitrile wash (to squeeze out the gel), another water wash and a final acetonitrile wash.
  • Samples can be desalted using, for example, 50 ⁇ l of activated AG-SO(H + ), filtered through a 0.45 ⁇ m LH Millipore filter and dried down for fluorescent labeling.
  • an in-gel-block release from protein mixtures can be used. Briefly, the whole protein mixture (e.g. serum or plasma) can be reduced and alkylated as in the In-gel oligosaccharide release described above, then set into 15% SDS-gel mixture but without bromophenol blue. A total volume of gel of 185 ⁇ l can be used (initially set into a 48 well plate, then removed for cutting up) with 300 ⁇ l of 100 U/ml of PNGaseF. The washing procedures can be similar to those used for in-gel-band release.
  • the whole protein mixture e.g. serum or plasma
  • a total volume of gel of 185 ⁇ l can be used (initially set into a 48 well plate, then removed for cutting up) with 300 ⁇ l of 100 U/ml of PNGaseF.
  • the washing procedures can be similar to those used for in-gel-band release.
  • This procedure can be more suitable for automated glycan release than in-solution PNGaseF release, and can be the preferred method for high throughput glycan analysis.
  • This system can be easily further modified to work with smaller amounts of gel set into a 96 well plate. Enzymatic release ofN-glycansfrom PVDF membranes
  • the glycoproteins in reduced and denatured serum samples can be attached to a hydrophobic PVDF membrane in a 96 well plate by simple filtration.
  • the samples can be then washed to remove contaminates, incubated with PNGaseF to release the glycans based on the methods described in Papac, D. L, et. al. Glycobiology 8: 445- 54, 1998, and in Callewaert, N., et. al. Electrophoresis 25: 3128-31, 2004, both incorporated hereby by reference in their entirety.
  • the iV-glycans can be then washed from the bound protein, collected and dried down ready for fluorescent labeling.
  • N- glycans can be released in situ from the glycoproteins by incubation with PNGaseF and by chemical means. Chemical release of N- and 0-glycans
  • Ammonia-based ⁇ -elimination can be used to release both N- and O-glycans by a modification of the classical ⁇ -elimination (Huang, Y. et. al. Analytical Chemistry 73: 6063-6069, 2001) which can be applied to glycoproteins in solution or on PVDF membranes. Ammonia-based ⁇ -elimination can be done from PVDF membranes. This strategy, can be optimized for high throughput, and can provide a powerful approach for releasing both N- and O-glycans in their correct molar proportions and in an open ring form suitable for post-release labeling.
  • Samples of glycoprotein, mixtures of glycoproteins, whole serum or other body fluids are reduced and alkylated by adding 4 ⁇ l of 5X sample buffer (5X sample buffer: 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water), 2 ⁇ l of 0.5M dithiothreitol (DTT) and water to make up to 20 ⁇ l in total, incubated at 7O 0 C for lOmin, then alkylated by addition of 2 ⁇ l of 10OmM iodoacetamide and incubated for 30 min in the dark at room temperature.
  • 5X sample buffer 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water
  • DTT dithiothreitol
  • Protein binding PVDF membranes (Durapore 13mm x 0.45 ⁇ m HVHP, Millipore) in Swinnex filter holders (Millipore) are pre-washed with 2 x 2.5 ml water using an all-polypropylene 2.5 ml syringe (Sigma), followed by a syringe full of air to remove most of the liquid from the membrane.
  • the reduced and alkylated sample is then applied directly to the membrane and left to bind for 5 min before washing by pushing through 2 x 2.5 ml water slowly with a syringe, followed by a syringe full of air to remove most of the liquid from the membrane.
  • the filter with the bound glycoprotein samples is then carefully removed from the filter holder and placed in a 1.5 ml screw capped polypropylene tube with a molded PTFE cap.
  • 1 ml of ammonium carbonate saturated 29.2% aqueous ammonium hydroxide, plus lOOmg ammonium carbonate is added to the tube. This is incubated for 40 hours at 6O 0 C, then cooled in the fridge. The liquid is then transferred to a clean tube and evaporated to dryness.
  • the released glycans are re-dissolved in water and re-dried until most of the salts are removed.
  • 100 ⁇ l of 0.5M boric acid is added to the glycans and incubated at 37 0 C for 30 min. The glycans are then dried under vacuum, ImI methanol added, re-dried, a further 1 ml methanol added and re-dried to remove the boric acid.
  • Labeling of glycans Upon releasing the glycans can be labeled with, for example, a fluorescent label or a radioactive label.
  • the fluorescent label can be, for example, 2- aminopyridine (2-AP), 2-aminobenzamide (2-AB), 2-aminoanthranilic acid (2-AA), 2-aminoacridone (AMAC) or 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS). Labeling of glycans with fluorescent labels is described, for example, by Bigge, J. C, et. al.
  • Fluorescent labels can label all glycans efficiently and non-selectively and can enable detection and quantification of glycans in the sub picomole range.
  • the choice of fluorescent label depends on the separation technique used. For example, a charged label is specifically required for capillary electrophoresis.
  • 2 AB label can be preferred for chromatographic, enzymatic and mass spectroscopic processes and analyses, while 2- AA label can be preferred for electrophoretic analyses.
  • Unlabelled glycans can be also detected by, for example, mass spectrometry, however, fluorescent labelling may aid glycan ionisation, see e.g. Harvey, D. J. (1999). "Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates.” Mass Spectrom Rev 18: 349-450.; Harvey, D. J. (2000). Electrospray mass spectrometry and fragmentation of N-linked carbohydrates derivatized at the reducing terminus. J Am Soc Mass Spectrom 11 : 900-915.
  • Quantitatively analyzing the glycans can be carried out, for example, by chromatography, mass spectrometry, electrophoresis or a combination thereof.
  • the chromatographic technique can be high performance anion exchange chromatography (HPAEC), weak ion exchange chromatography (WAX), gel permeation chromatography (GPC), high performance liquid chromatography (HPLC), normal phase high performance liquid chromatography (NP-HPLC), reverse phase HPLC (RP-HPLC), or porous graphite carbon HPLC (PGC-HPLC).
  • the mass spectrometry technique can be, for example, matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS), electrospray ionization time of flight mass spectrometry (ESI-TOF-MS), or liquid chromatography mass spectrometry (LC-MS).
  • the electrophoretic technique can be, for example, gel electrophoresis or capillary electrophoresis. The use of these separation techniques for analyzing glycans is described, for example, in the following publications:
  • Quantitatively analyzing the glycans can be determining particular glycan structures present in the released glycans.
  • the particular glycan structure can be detected down to subpicomolar levels.
  • the results of quantitatively analyzing the glycans can be presented as glycosylation profiles.
  • the glycosylation profiles can comprise a plurality of peaks corresponding to the glycan structures present in the released glycans.
  • glycans of glycoproteins can be released from two groups of samples: 1) samples from a body fluid of patients diagnosed with the autoimmune disease and 2) control samples.
  • Control samples can be samples of a body fluid of a single healthy patient or from a pool of healthy patients. Healthy in context of the previous sentence means not diagnosed with the autoimmune disease.
  • Quantitatively analyzing the glycans in this embodiment comprises quantitatively analyzing glycans released from the control samples and quantitatively analyzing glycans released from the diseased samples.
  • Quantitatively analyzing the glycans can further comprise comparing the glycan profiles of the diseased glycans and the control glycans to determine a glycosylation marker of the autoimmune disease.
  • comparing the glycan profiles of the diseased glycans and the control glycans to determine a glycosylation marker of the autoimmune disease can comprise comparing peak ratios in the diseased glycan profiles and in the controlled glycan profiles.
  • One or more of the peak ratios with the highest correlation with parameters of the diseased patents can be selected as the glycosylation marker of the autoimmune disease.
  • the parameters of the autoimmune disease patients can be, for example, diagnosis age, sex, disease stage, disease activity, disease severity, disease prognosis, remission, response to therapy or medication.
  • the glycosylation marker can be applied for diagnosing, monitoring, prognosticating the autoimmune disease or predicting a response to a specific therapy or medication in one or more new patients in a high throughput fashion.
  • Applying the glycosylation marker to diagnosing and monitoring the autoimmune disease in new patients can be carried out, for example, by releasing glycans of glycoproteins from a body fluid of a new patient, quantitatively analyzing the glycans from the new patient and determining a relative level of the glycosylation marker in the glycosylation profile of the glycans from the new patient.
  • One embodiment of the present invention is a method of diagnosing and monitoring an autoimmune disease comprising measuring diseased glycosylation profiles and one or more control glycosylation profiles, wherein the diseased glycosylation profiles are glycosylation profiles of glycans of glycoproteins from autoimmune disease patients and the one or more control glycosylation profiles are glycosylation profiles of glycans of glycoproteins from patients without the autoimmune disease; and comparing peak ratios in the diseased glycosylation profiles and in the one or more control glycosylation profiles and selecting a ratio having a highest correlation with parameters of the autoimmune disease patients out of the peak ratios as a glycosylation marker of the autoimmune disease.
  • Measuring the glycosylation profiles in this embodiment can be carried out by any of the described above methods. Most preferably, quantification of glycans is carried out by HPLC or by HPLC in combination with mass spectrometry.
  • both glycans from autoimmune disease patients and control glycans can be released without purifying the glycoproteins. Both glycans from autoimmune disease patients and control glycans can be also released from purified glycoproteins.
  • purified glycoproteins can be serum immunoglobulin G (IgG), serum immunoglobulin A (IgA), IgM, complement components, or inflammatory markers.
  • Glycans from autoimmune disease patients and control glycans can be also released from a body fluid without purifying the glycoproteins. Glycans can be released by the described above techniques.
  • the method of this embodiment can further comprise applying the glycosylation marker to diagnosing the autoimmune disease, monitoring the autoimmune disease, prognosticating the autoimmune disease, or. predicting a response to a therapy in one or more new patients.
  • Yet another embodiment of the present invention is a high throughput method for diagnosing and monitoring rheumatoid arthritis in a patient comprising releasing glycans of glycoproteins from a body fluid or a body tissue of the patient; and measuring galactosylation of the released glycans.
  • Measuring galactosylation can be, for example, measuring a ratio between an amount of GO glycans and an amount of Gl glycans in the glycans.
  • GO denotes glycans having no galactose
  • Gl denotes glycans comprising exactly one galactose.
  • Glycans can be released from a body fluid or body tissue of the patient without purifying the glycoproteins, without treating the glycans with exoglycosidase and without exposing the body fluid or the body tissue to hydrazinolysis. Glycans can be released, for example, using one of the described above methods. Analysis of glycans in this embodiment can be carried out using any of the mentioned above techniques but preferably by HPLC, mass spectrometry or a combination thereof. Upon the glycans release, glycans can be labeled with fluorescent or radioactive label as discussed in other embodiments of the invention.
  • the body fluid can be, for example, whole serum, blood plasma, synovial fluid, urine, seminal fluid, or saliva.
  • This study is used to demonstrate that a direct measurement of glycans released from whole serum can be used as marker for rheumatoid arthritis without IgG purification by correlating GO/triple-Gl ratio from undigested glycans released from whole serum with the amount of GO glycans as a percentage of the total glycans released from purified IgG. Selection of patient sample.
  • Control patient serum was pooled discarded clinical material from individuals undergoing routine employee health screening.
  • RA patients were selected based on a combination of physician global activity score, rheumatoid factor seropositivity and active joint count.
  • IgG purification :
  • IgG was isolated from whole serum via affinity chromatography employing protein-G sepharose as described in 'Antibodies: A laboratory manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988, and P.L. Ey et. al. "Isolation of pure IgG;, IgG 2a andIgG 2b immunoglobulins from mouse serum using protein A- Sepharose ", Molecular Immunology, vol. 15, pp. 429, 1978, both incorporated hereby by reference in their entirety. Briefly, lOO ⁇ l of whole serum was diluted with 300 ⁇ l of 10OmM Tris pH 8.0 and allowed to pass over a ImI column of protein-G sepharose beads (Amersham Biosciences).
  • IgG presence in eluted fractions was confirmed via 10% polyacryl amide gel electrophoresis (PAGE) under reducing conditions (as described, e.g., in Laemmli, "Cleavage of structural proteins during the assembly of the head of bacteriophage TY', Nature, : 227, 680-685, 1970, incorporated hereby by reference in its entirety) and via western blot (Current Protocols in Immunology. John Wiley and Sons, 1994, incorporated hereby by reference in its entirety) utilizing horseradish-peroxidase conjugated donkey-anti- human IgG (Jackson Immunochemicals) and visualized with Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer). Quantitative depletion of serum IgG in column flow through material was confirmed via western blot analysis. Glycans release:
  • Glycans were released from purified IgG by running the reduced and alkylated sample on sodium-dodecyl sulphate polyacryl amide gel electrophoresis (SDS- PAGE), cutting out the heavy chain and digesting with peptide N-glycosidase F (PNGaseF) as described in Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A., and Harvey, D. J. (1997). Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography.
  • PNGaseF peptide N-glycosidase F
  • Figure 1 shows SDS-PAGE and NP-HPLC profiles from samples GBRAl and GBRAl 3.
  • insets (a) and (b) of figure 1 provide SDS-PAGE gel pictures of the purified IgGs from the respective samples separated into heavy (H) and light (L) chain bands.
  • Insets (c) and (d) of figure 1 provide NP-HPLC profiles for heavy and light chain glycans released from the gel bands shown in (a) and (b) and not subjected to digestion with sialidase and fucosidase. Since no glycans were detected on the light chain, only the heavy chain was required for analysis.
  • Figure 2 illustrates the details of (a) the measurement of the GO/triple-Gl ratio directly from undigested glycans released from purified IgG and (b) the 'classic' measurement of the ratio GO glycans to the total glycans released from purified IgG and digested with sialidase and fucosidase.
  • Figure 2 shows NP-HPLC profiles from the sample GBRAl 5. Each peak corresponds to certain glycan(s). The peaks in each profile are integrated to give the area under the curve for each peak.
  • the ratio GO/triple-Gl is actually the peak area of FcA2G0 divided by the peak area of FcA2Gl[6]+FcA2Gl[3]+FcA2BGl[6] +FcA2BGl[3] (which elutes as a triplet).
  • the area under the peaks corresponding to the GO peaks is divided by the total area under all the peaks in the profile and expressed as a percentage.
  • Figure 3 illustrates NP-HPLC profiles of control sample and the sample GBRAl 5.
  • insets (a) and (d) show glycans released from whole sera of the respective samples
  • insets (b) and (e) show undigested heavy chain glycans released from respective purified IgGs
  • insets (c) and (f) show heavy chain glycans released from respective purified IgGs and digested with sialidase and fucosidase.
  • Table 1 lists the ratios of the GO to triple-Gl peak from whole serum and purified IgG from the same serum samples from 15 RA patients and one pooled control.

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Abstract

La présente invention concerne une technique permettant de diagnostiquer et de surveiller une maladie auto-immune qui consiste à libérer du glycane de glycoprotéines en provenance de liquides organiques sans purifier ces glycoprotéines et sans exposer les liquides organiques a une hydrazinolyse et, à analyser de manière quantitative ces glycanes une autre technique de diagnostic et de surveillance de maladie auto-immune consiste à mesurer des profils d'une de glycosylation de malade et un ou plusieurs profils de glycosylation témoin, les profils de glycosylation de malade étant des profils de glycosylation de glycanes de glycoprotéines provenant de patients atteint de maladie auto-immune et le ou les profils de glycosylation témoin étant des profils de glycosylation de glycanes de glycoprotéines de patients exempts de maladie auto-immune et, à comparer et des rapport de crêtes de dans les profils de glycosylation de malade et dans le ou les profils de glycosylation témoin et à sélectionner une partie possédant la plus forte corrélation avec des paramètres des patients atteints de maladie auto-immune en dehors des rapports de crêtes comme marqueur de glycosylation de cette maladie auto-immune. Une technique de haut rendement permettant de diagnostiquer de surveiller l'arthrite rhumatoïde chez un patient consiste à libérer des glycanes de glycoprotéines de liquides organiques ou de tissu organique du patient, à mesurer un rapport entre une quantité de G0 glycanes et une quantité de G1 glycanes dans ces glycanes, les g0 glycanes ne comprenant pas de galactose et les g1 glycane comprenant exactement un galactose.
PCT/IB2005/002885 2005-04-26 2005-06-24 Analyse de glycane a haut rendement pour diagnostiquer et surveiller l'arthrite rhumatoide et d'autres maladies auto-immunes WO2006114661A1 (fr)

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WO2009086952A2 (fr) * 2008-01-07 2009-07-16 Projech Science To Technology, S.L. Compositions de traitement de maladies articulaires dégénératives
EP2256499A1 (fr) * 2008-01-29 2010-12-01 National University Corporation Hokkaido University Procédé de diagnostic de polyarthrite rhumatoïde par analyse d'une chaîne de sucre
WO2012012725A2 (fr) 2010-07-23 2012-01-26 President And Fellows Of Harvard College Méthodes de dépistage de maladies ou d'affections à l'aide de cellules phagocytaires
WO2014040066A1 (fr) * 2012-09-10 2014-03-13 The Johns Hopkins University Analyse, en phase solide, de glycanes et de glycopeptides et puce microfluidique pour l'extraction et l'analyse glycomiques, et ses procédés d'utilisation
CN105277718A (zh) * 2015-09-29 2016-01-27 上海知先生物科技有限公司 用于恶性肿瘤相关筛查及评估的产品、应用及方法
CN107817353A (zh) * 2017-11-01 2018-03-20 复旦大学 预测肿瘤坏死因子抑制剂疗效的试剂和方法
US10494675B2 (en) 2013-03-09 2019-12-03 Cell Mdx, Llc Methods of detecting cancer
US10626464B2 (en) 2014-09-11 2020-04-21 Cell Mdx, Llc Methods of detecting prostate cancer
US10934589B2 (en) 2008-01-18 2021-03-02 President And Fellows Of Harvard College Methods of detecting signatures of disease or conditions in bodily fluids
US10961578B2 (en) 2010-07-23 2021-03-30 President And Fellows Of Harvard College Methods of detecting prenatal or pregnancy-related diseases or conditions
CN112924672A (zh) * 2021-01-28 2021-06-08 中国医学科学院北京协和医院 一种用于诊断类风湿关节炎合并肺间质纤维化的生物标志物及其用途
KR20210097866A (ko) * 2020-01-30 2021-08-10 충남대학교산학협력단 반려동물 골관절염 진단용 당사슬 바이오마커 및 이를 이용한 반려동물 골관절염 진단방법
US11111537B2 (en) 2010-07-23 2021-09-07 President And Fellows Of Harvard College Methods of detecting autoimmune or immune-related diseases or conditions
EP3812759A4 (fr) * 2018-06-20 2022-07-27 Tosoh Corporation Procédé de séparation d'anticorps, et procédé de test d'une maladie
US11585814B2 (en) 2013-03-09 2023-02-21 Immunis.Ai, Inc. Methods of detecting prostate cancer
EP4303584A2 (fr) 2010-07-23 2024-01-10 President and Fellows of Harvard College Procédés de détection de signatures de maladies ou pathologies dans des liquides biologiques

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WO2009086952A3 (fr) * 2008-01-07 2010-04-01 Projech Science To Technology, S.L. Compositions de traitement de maladies articulaires dégénératives
WO2009086952A2 (fr) * 2008-01-07 2009-07-16 Projech Science To Technology, S.L. Compositions de traitement de maladies articulaires dégénératives
US10934588B2 (en) 2008-01-18 2021-03-02 President And Fellows Of Harvard College Methods of detecting signatures of disease or conditions in bodily fluids
US11001894B2 (en) 2008-01-18 2021-05-11 President And Fellows Of Harvard College Methods of detecting signatures of disease or conditions in bodily fluids
US10934589B2 (en) 2008-01-18 2021-03-02 President And Fellows Of Harvard College Methods of detecting signatures of disease or conditions in bodily fluids
EP2256499A1 (fr) * 2008-01-29 2010-12-01 National University Corporation Hokkaido University Procédé de diagnostic de polyarthrite rhumatoïde par analyse d'une chaîne de sucre
EP2256499A4 (fr) * 2008-01-29 2012-01-25 Univ Hokkaido Nat Univ Corp Procédé de diagnostic de polyarthrite rhumatoïde par analyse d'une chaîne de sucre
EP2535719A3 (fr) * 2008-01-29 2013-01-16 National University Corporation Hokkaido University Procédé de diagnostic de polyarthrite rhumatoïde par analyse d'une chaîne de sucre
WO2012012725A2 (fr) 2010-07-23 2012-01-26 President And Fellows Of Harvard College Méthodes de dépistage de maladies ou d'affections à l'aide de cellules phagocytaires
US11111537B2 (en) 2010-07-23 2021-09-07 President And Fellows Of Harvard College Methods of detecting autoimmune or immune-related diseases or conditions
EP4303584A2 (fr) 2010-07-23 2024-01-10 President and Fellows of Harvard College Procédés de détection de signatures de maladies ou pathologies dans des liquides biologiques
US10961578B2 (en) 2010-07-23 2021-03-30 President And Fellows Of Harvard College Methods of detecting prenatal or pregnancy-related diseases or conditions
WO2014040066A1 (fr) * 2012-09-10 2014-03-13 The Johns Hopkins University Analyse, en phase solide, de glycanes et de glycopeptides et puce microfluidique pour l'extraction et l'analyse glycomiques, et ses procédés d'utilisation
US10494675B2 (en) 2013-03-09 2019-12-03 Cell Mdx, Llc Methods of detecting cancer
US11585814B2 (en) 2013-03-09 2023-02-21 Immunis.Ai, Inc. Methods of detecting prostate cancer
US10626464B2 (en) 2014-09-11 2020-04-21 Cell Mdx, Llc Methods of detecting prostate cancer
CN105277718A (zh) * 2015-09-29 2016-01-27 上海知先生物科技有限公司 用于恶性肿瘤相关筛查及评估的产品、应用及方法
CN107817353A (zh) * 2017-11-01 2018-03-20 复旦大学 预测肿瘤坏死因子抑制剂疗效的试剂和方法
EP3812759A4 (fr) * 2018-06-20 2022-07-27 Tosoh Corporation Procédé de séparation d'anticorps, et procédé de test d'une maladie
KR20210097866A (ko) * 2020-01-30 2021-08-10 충남대학교산학협력단 반려동물 골관절염 진단용 당사슬 바이오마커 및 이를 이용한 반려동물 골관절염 진단방법
KR102288646B1 (ko) 2020-01-30 2021-08-11 충남대학교산학협력단 반려동물 골관절염 진단용 당사슬 바이오마커 및 이를 이용한 반려동물 골관절염 진단방법
CN112924672A (zh) * 2021-01-28 2021-06-08 中国医学科学院北京协和医院 一种用于诊断类风湿关节炎合并肺间质纤维化的生物标志物及其用途

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