Patent application title: BIOMARKER FOR OSTEOARTHRITIS AND/OR OTHER AGEING-RELATED DISEASES, AND USE THEREOF
Inventors:
Yves Henrotin (Beaufays, BE)
Myriam Gharbi (Liege, BE)
Michelle Deberg (Embourg, BE)
Edwin De Pauw (Marchin, BE)
IPC8 Class: AG01N33566FI
USPC Class:
436501
Class name: Chemistry: analytical and immunological testing biospecific ligand binding assay
Publication date: 2011-06-30
Patent application number: 20110159607
Abstract:
The invention relates to the identification of a biomarker whose
abundance in biological samples is changed in subjects with
osteoarthritis and/or other ageing-related diseases. The biomarker has
applications in the diagnosis of osteoarthritis and/or other
ageing-related diseases, in determining the prognosis for an individual
diagnosed with osteoarthritis and/or other ageing-related diseases, and
in monitoring the efficacy of treatment for osteoarthritis and/or other
ageing-related diseases.Claims:
1. A method of determining the osteoarthritis status of a subject, and/or
the status of another ageing-related disease in a subject, comprising the
steps of: (i) determining the concentration in a biological sample of a
peptide comprising the same or substantially the same amino acid sequence
as Seq ID no:1 or a fragment thereof; (ii) comparing the peptide
concentration determined in step (i) with one or more reference values.
2. A method of diagnosing osteoarthritis and/or another ageing-related disease in a subject, comprising the steps of: (i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof; (ii) comparing the peptide concentration determined in step (i) with one or more reference values.
3. A method of determining the prognosis for a subject with osteoarthritis and/or another ageing-related disease, comprising the steps of: (i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof; (ii) comparing the peptide concentration determined in step (i) with one or more reference values.
4. A method of determining the efficacy of a treatment for osteoarthritis and/or another ageing-related disease in a subject, comprising the steps of: (i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof; (ii) administering a treatment for osteoarthritis and/or another ageing related disease to the subject; (iii) determining after treatment the concentration in another biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof; (iv) comparing the peptide concentrations determined in step (i) and (iii) with one another, and optionally with one or more reference values.
5. The method of claim 1 wherein the biological sample is selected from the group comprising urine, whole blood, blood serum, blood plasma, synovium, sweat, cerebrospinal fluid, mucous, saliva, lymph, bronchial aspirates and milk.
6. The method of claim 1 wherein the concentration of the peptide is determined by using an immunoassay.
7. The method of claim 1 wherein the reference value is the concentration of the peptide in biological samples of one or more normal subjects.
8. A kit for use in determining the osteoarthritis status, or the status of another ageing-related disease, in a subject comprising at least one agent for determining the concentration of a peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof, in a biological sample.
9. The kit according to claim 8 wherein the agent is selected from the group comprising an enzyme, an antibody, a protein probe, a metabolite or any other suitable composition.
10. The kit according to claim 8 wherein the agent comprises two antibodies directed to different epitopes on the peptide.
11. The kit according to claim 10 for use in determining the concentration of the peptide by using a sandwich immunoassay.
12. An isolated peptide which comprises the same or substantially the same amino acid sequence as the amino acid sequence of Seq ID NO: 1, or a part thereof, or its amide, or a salt thereof.
13. The polynucleotide encoding a peptide according to claim 12.
14. The recombinant vector comprising a polynucleotide according to claim 13.
15. The transformant transformed with a recombinant vector according to claim 14.
16. The of a peptide according to claim 12 in the manufacture of an antibody.
17. The antibody specific for a peptide according to claim 12.
Description:
[0001] The present invention relates to a biomarker for osteoarthritis
and/or other ageing-related diseases. In particular, the invention
relates to a method of diagnosing osteoarthritis, and/or other
ageing-related diseases, by determining the level of a biomarker in a
biological sample. The invention also relates to the use of a biomarker
found in biological samples to monitor the efficacy of a treatment for
osteoarthritis, and/or other ageing-related diseases, and to determine
the prognosis for an individual diagnosed with osteoarthritis, and/or
another ageing-related disease.
[0002] Osteoarthritis is a progressive disorder characterized by destruction of articular cartilage and subchondral bone, and by synovial changes. Currently the diagnosis of osteoarthritis is based on clinical and radiographic changes which occur late during disease progression. More specifically, diagnosis is based on cartilage integrity, which as articular cartilage is invisible on radiographs must be assessed indirectly from the spacing between subchondral bone ends in a joint. This method does not allow detection of early structural damage, and is cumbersome to use in daily practice.
[0003] Biochemical markers of bone, synovium or cartilage turnover have been proposed as potential tools for the diagnosis, prognosis and treatment monitoring of osteoarthritis (Garnero, P., et al., Ann Rheum Dis, 2001. 60 (6): p. 619-26; Bruyere, O., et al., J Rheumatol, 2003. 30 (5): p. 1043-50; and Wu, J., et al., Arthritis Rheum, 2007. 56 (11): p. 3675-84). More specifically, Wu, J., et al. (Arthritis Rheum, 2007. 56 (11): p. 3675-84) describe potential molecular mediators and biomarkers of osteoarthritis in cartilage tissue. The method used required articular cartilage to be obtained, which requires an invasive procedure and provides only a limited amount of tissue. The method is therefore costly, time consuming and unsuitable for routine diagnostic testing, or for monitoring disease progression, or for determining the therapeutic effect of a treatment.
[0004] There therefore remains a need for a simple, rapid and effective method for the diagnosis of osteoarthritis and/or other ageing-related diseases, and/or to monitor the efficacy of treatments for osteoarthritis and/or other ageing-related diseases, and/or to determine the prognosis for a patient diagnosed with osteoarthritis and/or other ageing-related diseases.
[0005] The present invention provides a method for (i) diagnosing osteoarthritis and/or another ageing-related disease, (ii) determining the prognosis for a patient with osteoarthritis and/or another ageing-related disease, and (iii) monitoring the efficacy of treatment for osteoarthritis and/or another ageing-related disease, using readily availables which are simple to obtain and allow for rapid and cost effective use.
[0006] Reference herein to ageing-related diseases includes osteoporosis, and other degenerative diseases.
[0007] According to one aspect, the present invention provides a method of determining the osteoarthritis status of a subject, and/or the status of another ageing-related disease in a subject, comprising the steps of: [0008] (i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof; [0009] (ii) comparing the peptide concentration determined in step (i) with one or more reference values.
[0010] According to another aspect, the present invention provides a method of diagnosing osteoarthritis and/or another ageing-related disease in a subject, comprising the steps of: [0011] (i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof; [0012] (ii) comparing the peptide concentration determined in step (i) with one or more reference values.
[0013] According to yet another aspect, the present invention provides a method of determining the prognosis for a subject with osteoarthritis and/or another ageing-related disease, comprising the steps of: [0014] (i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof; [0015] (ii) comparing the peptide levels determined in step (i) with one or more reference values.
[0016] According to a further aspect, the present invention provides a method of determining the efficacy of a treatment for osteoarthritis and/or another ageing-related disease in a subject, comprising the steps of: [0017] (i) determining the concentration in a biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof; [0018] (ii) administering a treatment for osteoarthritis and/or another ageing related disease to the subject; [0019] (iii) determining after treatment, the concentration in another biological sample of a peptide comprising the same or substantially the same amino acid sequence as Seq ID no:1 or a fragment thereof; [0020] (iv) comparing the peptide concentrations determined in step (i) and (iii) with one another, and optionally with one or more reference values.
[0021] Preferably, in any method of the invention, the concentration of a peptide with the same sequence as the sequence of Seq ID no: 1 is determined. Alternatively, the concentration of a peptide substantially the same as the sequence of Seq ID no: 1 may be determined.
[0022] Alternatively, the concentration of a peptide fragment having a sequence the same, or substantially the same, as part of the sequence of Seq ID no: 1 may be determined. Preferably if the concentration of a fragment having a sequence the same, or substantially the same, as part of the sequence of Seq ID no: 1 is determined, the fragment represents an epitope within the sequence of Seq ID no: 1. Preferably the peptide fragment is at least 10, preferably at least 20, more preferably at least 30 amino acids long.
[0023] Alternatively to a peptide or a peptide fragment one may also consider a free fragment which is intended to refer to a polypeptide, a peptide or otherwise released from mammalian serpine B1 molecule by an oxidative or enzymatic processing. A free fragment is different from a native protein by its structure and configuration and may undergo modification such as phosphorylation, glycosylation or any other post-traductional modification resulting of a pathological mechanism. The free fragment as well as the peptide or peptide fragment contributes to the identification of the pathologic status of osteoarthritis patient.
[0024] An epitope is a binding site of an antibody on an antigen. In a peptide antigen, generally a linear epitope will be at least about 7 amino acids in length, and may be at least 8, at least 9, at least 10, at least 11, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, or at least 30 amino acid residues in length. However, antibodies may also recognise conformational determinants formed by non-contiguous residues on an antigen, and an epitope can therefore require a larger fragment of the antigen to be present for binding, e.g. a domain.
[0025] Reference herein to "a sequence substantially the same as" all or part of the sequence of Seq ID no: 1, refers to a peptide with a sequence which has at least 80%, preferably at least 90%, more preferably at least 95% or 98%, sequence identity with all or part of the sequence of Seq ID No:1. Preferably the sequence is the same or substantially the same as at least about 10, 15, 20, 25, 30, 35, 40, 45 or more consecutive amino acids on the sequence of Seq ID No:1
[0026] Homology or sequence identity of two or more amino acid sequences can be measured by using a homology scoring algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool). Alternatively, the UWGCC Package provides the BESTFIT program which can be used to calculate sequence identity between two or more sequences (e.g. used on its default setting) (Devereux et al (1984) Nucleic Acids Research 12 p 387-395).
[0027] The sequence of Seq ID no:1 represents a fragment of the serpin B1 protein. The fragment may be a degradation product of serpin B1.
[0028] In any method of the invention the peptide comprising a sequence the same or substantially the same as the sequence of Seq ID no: 1 or a part thereof is preferably differentially present in the sample from a subject with osteoarthritis or another ageing-related disease compared to a normal subject.
[0029] A peptide comprising the same or substantially the same sequence as Seq ID no: 1 or a fragment thereof, which is measured in step (i) and/or step (iii) in any method of the invention, is also referred to herein as the biomarker or the biomarker peptide.
[0030] Reference to the "osteoarthritis status" or to the "status of an ageing-related disease" refers to any distinguishable manifestation of osteoarthritis or the ageing-related disease, including diseased and non-diseased. For example, osteoarthritis status includes, without limitation, the presence or absence of osteoarthritis in a subject, the risk of a subject developing osteoarthritis, the stage of the disease, the progression of the disease, and the effectiveness or response of a subject to treatment for osteoarthritis.
[0031] Any method of the invention may be used in conjunction with the assessment of clinical symptoms and/or imaging results and/or the concentration of one or more other biomarkers.
[0032] Preferably all methods of the invention are carried out in vitro.
[0033] The biological sample may comprise urine, whole blood, blood serum, blood plasma, synovium, sweat, cerebrospinal fluid, mucous, saliva, lymph, bronchial aspirates, milk and the like. Preferably the biological sample is urine.
[0034] Biological sample, such as urine, have the advantage of being abundant and easily accessible.
[0035] A further advantage of using a biological sample, compared to a tissue such as cartilage, is that the progression of osteoarthritis or another ageing-related disease, and/or the therapeutic effect of a treatment, may be monitored by taking and testing samples as often as necessary without the need for invasive procedures.
[0036] The concentration of the biomarker peptide in a sample may be determined by any suitable assay. A suitable assay may include one or more of the following methods, an enzyme assay, an immunoassay, mass spectrometry, HPLC, electrophoresis or an antibody microarray, or any combination thereof. If an immunoassay is used it may be an enzyme linked immunoassay (ELISA), a sandwich assay, a competitive assay, a radioimmunoassay, a Western Blot, an immunoassay using a biosensor, an immunoprecipitation assay, an agglutination assay, a turbidity assay or a nephlelometric assay. If mass spectrometry is used it may be Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry.
[0037] Preferably the concentration of the biomarker peptide is determined using an immunoassay which uses one or more antibodies directed to the specific biomarker peptide to determine the concentration of the biomarker peptide in the sample.
[0038] If one or more antibodies are used to determine the concentration of a biomarker peptide in a sample the one or more antibodies may be synthetic, monoclonal, polyclonal, oligoclonal, bispecific, chimeric and/or humanised.
[0039] One or more of the antibodies used may comprise a tag or a label. The tag or label may be selected from the group comprising a radioactive, a fluorescent, a chemiluminescent, a dye, an enzyme, or a histidine tag or label, or any other suitable label or tag known in the art.
[0040] Preferably the reference value, to which the determined concentration of the biomarker peptide is compared, is the concentration of the same peptide in one or more "normal" subjects that do not have any detectable osteoarthritis and/or other ageing-related disease, or any clinical symptoms of osteoarthritis and/or other ageing-related disease, and have so called "normal values" of the biomarker peptide.
[0041] Alternatively, the reference value may be a previous value for the biomarker peptide obtained from a specific subject. This kind of reference value may be used if the method is to be used to monitor progression of osteoarthritis and/or another ageing-related disease, or to monitor the response of a subject to a particular treatment.
[0042] When the determined concentration of the biomarker is compared with a reference value, an increase or a decrease in the concentration of the biomarker may be indicative of the osteoarthritis status, and/or the status of another ageing-related disease, in the subject.
[0043] More specifically an increase or a decrease in the concentration of the biomarker may be indicative, or diagnostic, of osteoarthritis in the subject. Preferably, a decrease in the concentration of the biomarker, preferably of a peptide with the sequence of Seq ID no:1, in a sample is diagnostic of osteoarthritis.
[0044] Preferably an at least about 5 fold or more increase in the concentration of a peptide with the sequence of Seq ID no:1, or sequence substantially the same as Seq ID no:1, or a fragment thereof, in a sample compared to a reference sample from a normal subject is diagnostic of osteoarthritis. Preferably, an at least about 5.8 fold, decrease in the concentration of a peptide with the sequence of Seq ID no: 1, or sequence substantially the same as Seq ID no: 1, or a fragment thereof, in a sample compared to a reference sample from a normal subject is diagnostic of osteoarthritis.
[0045] The method of the invention may also be used to monitor osteoarthritis progression, and/or the progression of another ageing-related disease, in a subject. Furthermore, the method of the invention may be used to monitor the efficacy of a treatment for osteoarthritis, and/or another ageing-related disease, following administration of the treatment to a subject. Efficacy of a treatment may be monitored by analysing samples taken from a subject at various time points following initiation of the treatment. By monitoring changes in the concentration of the biomarker peptide over time and comparing these concentrations to normal and/or reference values, efficacy of the treatment may be determined.
[0046] In this case reference concentrations may include the initial concentration of the biomarker peptide in the subject, or the concentration of the biomarker peptide in the subject when they were last tested, or both.
[0047] According to another aspect of the invention there is provided a kit for use in determining the osteoarthritis status, or the status of another ageing-related disease, in a subject comprising at least one agent for determining the concentration of a peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No: 1, or a part thereof, in a sample provided by the subject.
[0048] The kit may be used to diagnose osteoarthritis and/or another ageing-related disease in a subject. The kit may alternatively be used to monitor disease progression or the efficacy of a treatment administered to a subject with osteoarthritis and/or another ageing-related disease.
[0049] The agent may be an enzyme, an antibody, a protein probe, a metabolite or any other suitable composition.
[0050] The agent for determining the concentration of one or more biomarker proteins is preferably labelled. The kit may also comprise means for detecting the label.
[0051] The kit may comprise one or more capture agents for capturing the peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof, in a sample provided by the subject. The capture agent may be one or more antibodies. The capture agent may be an antibody according to an aspect of the invention.
[0052] The kit may comprise two antibodies for use in a sandwich assay to determine the concentration of a peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof. Preferably the kit comprises two antibodies, each directed to a different epitope on the peptide comprising the same or substantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof. One antibody is preferably the capture antibody, and the other may comprise a label to allow its detection.
[0053] The capture agent may be attached to a solid support. The solid support may be a chip, a microtitre plate, a bead or a resin.
[0054] The kit may comprise instructions for suitable operational parameters in the form of a label or separate insert. The instructions may inform a user about how to collect the sample, and/or how to wash the capture agent.
[0055] The kit may comprise samples of the biomarker peptide to be detected. The samples of the biomarker peptide may be used as a standard for calibration and comparison. The kit may also comprise instructions to compare the concentration of the biomarker peptide detected in a sample with a calibration sample or chart. The kit may also include instructions indicating what concentration of the biomarker peptide is diagnostic of osteoarthritis and/or another ageing-related disease.
[0056] According to a yet further aspect, the invention provides the use of the determination of the concentration of the biomarker peptide in a biological sample of as a means of assessing the osteoarthritis status in a subject or as a means of assessing the status of another ageing-related disease in a subject.
[0057] According to a yet further aspect, the present invention provides the use of a biological sample, such as urine, as a source of at least one biomarker for the prognosis of osteoarthritis progression and/or another ageing-related disease, for diagnosing osteoarthritis and/or another ageing-related disease and for monitoring the effect of a treatment for osteoarthritis and/or another ageing-related disease.
[0058] According to another aspect the invention provides a peptide, which comprises the same or substantially the same amino acid sequence as the amino acid sequence of Seq ID NO: 1, or a part thereof, or its amide, or a salt thereof.
[0059] Preferably the peptide of the invention has a sequence the same as that of Seq ID No: 1.
[0060] According to a further aspect, the invention provides a polynucleotide encoding a peptide according to the invention. Preferably, the polynucleotide is a DNA molecule.
[0061] According to a yet further aspect the invention provides a recombinant vector, which comprises a polynucleotide of the invention.
[0062] According to another aspect the invention provides transformant, which is transformed with the recombinant vector of the invention.
[0063] In a further aspect, the invention provides the use of a peptide according to the invention in the manufacture of an antibody.
[0064] It a yet further aspect, the invention provides an antibody specific for a peptide according to the invention. In particular, the invention provides an antibody specific for a peptide having the sequence of Seq ID No: 1.
[0065] An antibody according to the invention may be synthetic, monoclonal, polyclonal, oligoclonal, bispecific, chimeric or humanised. The antibody may be complete or a fragment thereof, such as, Fv, Fab and F(ab)2 fragments. Methods of generating antibodies are well known in the art, and may include immunisation of suitable animals, such as, a rabbit, mouse, sheep or goat, with the peptide of interest (or an immunogenic fragment thereof) or recombinant techniques.
[0066] The skilled man will appreciate that preferred features of any one embodiment and/or aspect of the invention may be applied to all other embodiments and/or aspects of the invention.
[0067] Embodiments of the invention will now be described merely by way of example with reference to the accompanying figures in which:
[0068] FIG. 1A--shows an enlargement of a portion of a 2D-DIGE map from osteoarthritis (OA) subject (right) and non-osteoarthritis (NO) subject (left).
[0069] FIG. 1B--shows a representation of spot 351 volume variation between OA subjects (right) and non-osteoarthritis subjects (left).
[0070] FIG. 2--shows a graphic view of serpin B1 abundance modification based on the spot volume decrease shown in FIG. 1B.
[0071] FIG. 3A--shows the result of a mass spectrometry analysis of tryptic fragments of the peptide of serpin B1 recovered from spot 351 detailed in FIG. 1A, FIG. 1B and FIG. 2.
[0072] FIG. 3B--shows the sequence of human serpin B1 containing the fragments shown in FIG. 3A. (SEQ ID NO 3)
[0073] FIG. 4--shows the protein sequence of SEQ ID No. 1.
[0074] FIG. 5--shows the protein sequence of SEQ ID No. 2.
[0075] 2D-DIGE (two dimensional difference gel electrophoresis--Marouga et al, (2005) Anal Bioanal Chem 382 (3): 669-78) methodology is a powerful tool for investigating protein expression profiles in multiple sets of samples.
[0076] In this example, 2D-DIGE was used to study the protein expression profiles in urine samples from subjects with serious osteoarthritis and from healthy young subjects. Proteins in the samples to be compared were labelled with either Cy3 or Cy5 CyDye DIGE Fluors. The Cy2 CyDye DIGE Fluor was used to label a pooled sample comprising equal amounts of each of the samples within the study, and this used as an internal standard. The use of this internal standard ensured that all proteins present in the samples were represented, assisting both inter- and intra-gel matching.
Materials and Methods
Urine Samples Preparation
[0077] Urine samples were collected from 10 women undergoing hip replacement surgery due to severe osteoarthritis. Control samples were obtained from 5 healthy women (25.6±2.6 years) without articular degeneration. The urine samples were concentrated 100× by ultracentrifugation on Amicon Ultra-15 (Millipore, USA). Proteins were purified by precipitation using PlusOne 2D Clean-up kit (GE Healthcare, Sweden). Albumin depletion from urine samples was performed using affinity columns according to the Montage Albumin Deplete Kit (Millipore, USA) manual utilisation.
Labelling of Proteins with Cy3 and Cy5 Dyes
[0078] In all experiments, the purified proteins were labelled on lysine residues with Cy3 or Cy5 CyDye DIGE Fluors. The samples were minimal labelled which means that the ratio of dye to protein used was such that each protein molecule was labelled with only one dye molecule. Three gels were made as shown in Table 1. Proteins from different samples were labelled with Cy3 or Cy5 and loaded on the same gel. On the first and second gels, proteins from NO (normal) samples were labelled with Cy3 CyDye DIGE Fluor whereas proteins from osteoarthritis (OA) samples were labelled with Cy5 CyDye DIGE Fluor. Conversely, proteins from NO samples were labelled with Cy5 CyDye DIGE Fluor and proteins from OA samples were labelled with Cy3 CyDye DIGE Fluor on the third gel. An internal standard (MIX) comprising equal amounts of NO and OA samples was labelled with Cy2 CyDye DIGE Fluor and loaded on each gel.
TABLE-US-00001 TABLE 1 Gel 1 Gel 2 Gel 3 Cy3 NO NO OA Cy5 OA OA NO Cy2 MIX MIX MIX
[0079] In Table 1 OA means samples from subject with osteoarthritis, and NO means control samples from normal subjects.
Two-Dimensional Electrophoresis
[0080] Protein samples (37.5 μg) labelled with Cy3, Cy5 or Cy2 DIGE Fluor were separated by 2D electrophoresis using an IEF (ioselectric focusing) buffer (8 M urea, 2% CHAPS, 0.5% immobilized pH gradient [IPG] buffer, 1% DTT, and trace of bromophenol blue) which was loaded into an immobiline DryStrip (pH 3-10 NL, 24 cm) (GE Healthcare, Sweden). The first dimension isoelectric focusing was performed for 70,000 Vhr using a Protean IEF Cell (Biorad) at 20° C. Next, the gels were equilibrated for 12 minutes in equilibration buffer I (375 mM Tris-Cl [pH 8.8], 6 M urea, 20% glycerol, 2% SDS, and 130 mM DTT) and II (375 mM Tris-Cl [pH 8.8], 6 M urea, 20% glycerol, 2% SDS, and 135 mM IAA). The second dimension was run according to the Ettan DALTsix Electrophoresis Unit operating manual (GE Healthcare, Sweden). A 12.5% SDS-polyacrylamide slab gel (24 cm) was used for the second-dimension gel electrophoresis. The IPG strips were placed on the surface of the second-dimension gel. The gels were then placed in SDS electrophoresis buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS) and run overnight at 1.5 W per gel.
[0081] Gels were scanned while still between two low-fluorescence glass plates using a Typhoon 9400 fluorescent scanner and saved in .gel format using ImageQuant software (GE Healthcare, Sweden). The excitation wavelengths for Cy3 and Cy5 are 550 nm and 645 nm, and the emission wavelengths are 570 nm and 670 nm for Cy3 and Cy5, respectively. The excitation/emission wavelength of Cy2 is around 489/505 nm. Image analysis was performed on DeCyder® software (GE Healthcare, Sweden). Interesting spots with differential fluorescent intensity between Cy3 and Cy5 were picked out the gel in order to allow protein identification after post-staining with Coomassie Blue.
[0082] DeCyder 2D v6.5 software (GE Healthcare, Sweden) was used for simultaneous comparison of abundance changes across sample groups. The DeCyder differential in-gel analysis (DIA) module generated ratios for each protein "spot" by comparing Cy3 and Cy5 signals to the Cy2 control signal. The DeCyder biological variation analysis module matched all protein spot maps from the gels and normalized the DIA-generated Cy3:Cy2 and Cy5:Cy2 ratios relative to the Cy2 signals for each resolved feature separately. This enabled the calculation of average abundance changes across all three samples within each test group, and the application of univariate statistical analyses (Student's t-test, ANOVA).
Proteins Identification
[0083] Protein spots were cut out of the polyacrylamide gel and washed twice for 5 minutes with an ammonium hydrogenocarbonate (50 mM)-acetonitrile mix (1:1). Gel spots were incubated in dithiothreitol (10 mM), NH4HCO3 (50 mM), for 40 min in a 56° C. water bath. Proteins in the gel spots were alkylated for 1 h in the dark with iodoacetamide (55 mM) in NH4HCO3 (50 mM). The gel spots were then washed twice with an ammonium hydrogenocarbonate (50 mM)-acetonitrile mix (1:1), dehydrated with acetonitrile, and then dried for 15 minutes at room temperature. The gel spots were then rehydrated for 10 minutes on ice with modified trypsin (10 ng/μl) in NH4HCO3 (25 mM) and then incubated overnight at 37° C. Hydrolysis of peptides was stopped in TFA (1%)-ACN (5%) solution. The gel spots were then sonicated twice for 1 minute in order to release protein fragments out of the isolated gel spots. Protein fragments in solution were freeze-dried. The identity of proteins was determined by tandem mass spectrometry (MS-MS spectrometry). The Mowse score (Pappin et al (1993) Curr Biol June 1; 3 (6):327-32) gave the fidelity of identification.
Results
[0084] Proteins isolated from the urine samples and labelled with Cy3 or Cy5 were separated by two-dimensional electrophoresis. The first separation was performed with an isoelectric focusing range of pH 3-10 NL and a load of 37.5 μg of protein. 372 spots of proteins were matched between all gels. Spots with a modification of intensity between OA and NO with a ratio superior to 1.5 (t-test: p<0.05) were selected for protein identification using mass spectrometry. Table 2 shows the results of analysis of these spots, and details the size of the spot, the Mowse score (which is -10 log (P) where P is the probability that the observed match is a random event), the abundance ratio, the name of the protein identified in the spot and the accession number for the identified protein in the Swiss Prot database.
TABLE-US-00002 TABLE 2 Abun- dance Sequence ratio Spot coverage Mowse (OA/ Swiss-Prot no (%) score NO) Protein description Accession 40 9 390 1.83 Poly-Ig receptor P01833 (PIGR) 43 5 159 1.6 Poly-Ig receptor P01833 (PIGR) 75 10 340 -1.68 Transferrin P02787 219 11 334 -1.7 Kininogen-1 precursor P01042 6 55 Alpha 1 anti-trypsin P01009 (A1AT) 222 15 349 -1.64 Kininogen-1 precursor P01042 7 99 A1AT P01009 223 13 405 -1.89 Kininogen-1 precursor P01042 16 311 A1AT P01009 226 18 334 -1.73 A1AT P01009 6 219 Kininogen-1 precursor P01042 262 13 368 -1.91 Kininogen-1 precursor P01042 6 61 A1AT P01009 267 10 304 -1.98 Kininogen-1 precursor P01042 269 5 143 -1.83 Kininogen-1 precursor P01042 343 189 4.18 Beta-actine 348 21 192 2 Zn-α-2-glycoprotein P25311 precursor 349 8 195 -2.44 Serpin B3 P29508 351 6 115 -5.84 Serpin B1 P30740 352 5 130 2.2 Fibulin-3 Q12805 73 Apoptosis-inducing Q9BRQ8 factor 2 (two proteins identified in the same spot) 356 8 110 2.01 Zn-α-2-glycoprotein P25311 2 45 precursor Q12805 FIBULIN3 386 10 197 1.54 GP36b Q12907 398 5 187 2.28 AMBP protein P02760 precursor 485 8 289 -2.3 Mannan-binding lectin O00187 serine protease 2 E.C precursor 3.4.21.104
[0085] As can be seen from Table 2, various proteins with significant changes in concentration in samples from osteoarthritis compared to samples from normal subjects were identified. Some of the proteins identified are implicated in the inflammatory process, for example, the kininogen precursor or alpha-1-antitrypsin. This observation coincides with the pathology of osteoarthritis.
[0086] A significant decrease in the concentration of a specific serpin B1 fragment was observed in osteoarthritis subjects compared to normal subject, as shown in Table 2 and FIGS. 1A and 1B. In FIG. 1A there is shown an enlargement of the area around spot 351 on the 2D-DIGE map of proteins extracted from urine from osteoarthritis subjects (right) and a NO subjects (left). In FIG. 1B the same spot (spot 351) is represented by volume variation of the spot between samples from OA subjects (right) and NO subjects (left). A graphic view of the abundance modification based on the spot 351 volume decrease is also shown in FIG. 2.
[0087] Tryptic fragments from spot 351 were identified by mass spectrometry analysis as shown in FIG. 3A. These fragments were identified as being fragments from the protein serpin B1, as shown in FIG. 3B. FIG. 3B shows the protein sequence derived by the translation of the mRNA of human serpin B1 and in bold the specific sequence identified by mass spectrometry. Each tryptic fragment studied was given a score which is -10 log(P) where P is the probability that the observed match is a random event. Individual ion scores >26 indicate identity or extensive homology.
[0088] The serpins are a superfamily of serine protease inhibitors (SERine Protease Inhibitors). In human, serpin family is divided, into nine clades (A-I). These proteins are involved in a diverse set of processes including blood coagulation, inflammation and complement activation, cell migration, pro-hormone activation and matrix remodelling. They undergo a unique and dramatic conformational change when they inhibit target proteases. Serpines inhibit protease with substrate-like irreversible inhibitory mechanism. Not all serpins functions as proteinase inhibitors. Some of them act as chaperone proteins (heat shock protein 47) or are involved in blood pressure regulation (angiotensinogen).
[0089] Clade B represents a particular set of serpines, ov-serpines, with amino acid similarities among chicken ovalbumin. Most serpins target serine protease, however, some serpines from clade B can inhibit serine or cysteine protease or both.
[0090] The serpin B1 or LEI (leukocyte elastase inhibitor) is involved in regulating the degradation of the extracellular matrix through its action on neutrophil elastase. The elastase is also involved in the pathogenesis of inflammatory diseases such as rheumatoid arthritis and has been proposed as biomarker of chronic joint diseases (Kleesiek et al., 1986; Momohara et al, 1997). In the lung, administration of recombinant serpin B1 has a protective role against proteases released by neutrophils (Rees et al. 1999, Hirche et al., 2004; Rubio et al., 2004; and Yasumaki al., 2006).
[0091] An increased amount of serpin in plasma may be associated with a particular pathology. For example, serpin B3 is associated with squamous cell carcinoma and serves as a diagnostic marker for some cancers (Pemberton et al., 1997).
Sequence CWU
1
31140PRTHomo sapiens 1Glu Ala Thr Thr Asn Ala Pro Phe Arg Leu Asn Lys Lys
Asp Arg Lys1 5 10 15Thr
Val Lys Met Met Tyr Gln Lys Lys Lys Phe Ala Tyr Gly Tyr Ile 20
25 30Glu Asp Leu Lys Cys Arg Val Leu
Glu Leu Pro Tyr Gln Gly Glu Glu 35 40
45Leu Ser Met Val Ile Leu Leu Pro Asp Asp Ile Glu Asp Glu Ser Thr
50 55 60Gly Leu Lys Lys Ile Glu Glu Gln
Leu Thr Leu Glu Lys Leu His Glu65 70 75
80Trp Thr Lys Pro Glu Asn Leu Asp Phe Ile Glu Val Asn
Val Ser Leu 85 90 95Pro
Arg Phe Lys Leu Glu Glu Ser Tyr Thr Leu Asn Ser Asp Leu Ala
100 105 110Arg Leu Gly Val Gln Asp Leu
Phe Asn Ser Ser Lys Ala Asp Leu Ser 115 120
125Gly Met Ser Gly Ala Arg Asp Ile Phe Ile Ser Lys 130
135 1402379PRTHomo sapiens 2Met Glu Gln Leu Ser
Ser Ala Asn Thr Arg Phe Ala Leu Asp Leu Phe1 5
10 15Leu Ala Leu Ser Glu Asn Asn Pro Ala Gly Asn
Ile Phe Ile Ser Pro 20 25
30Phe Ser Ile Ser Ser Ala Met Ala Met Val Phe Leu Gly Thr Arg Gly
35 40 45Asn Thr Ala Ala Gln Leu Ser Lys
Thr Phe His Phe Asn Thr Val Glu 50 55
60Glu Val His Ser Arg Phe Gln Ser Leu Asn Ala Asp Ile Asn Lys Arg65
70 75 80Gly Ala Ser Tyr Ile
Leu Lys Leu Ala Asn Arg Leu Tyr Gly Glu Lys 85
90 95Thr Tyr Asn Phe Leu Pro Glu Phe Leu Val Ser
Thr Gln Lys Thr Tyr 100 105
110Gly Ala Asp Leu Ala Ser Val Asp Phe Gln His Ala Ser Glu Asp Ala
115 120 125Arg Lys Thr Ile Asn Gln Trp
Val Lys Gly Gln Thr Glu Gly Lys Ile 130 135
140Pro Glu Leu Leu Ala Ser Gly Met Val Asp Asn Met Thr Lys Leu
Val145 150 155 160Leu Val
Asn Ala Ile Tyr Phe Lys Gly Asn Trp Lys Asp Lys Phe Met
165 170 175Lys Glu Ala Thr Thr Asn Ala
Pro Phe Arg Leu Asn Lys Lys Asp Arg 180 185
190Lys Thr Val Lys Met Met Tyr Gln Lys Lys Lys Phe Ala Tyr
Gly Tyr 195 200 205Ile Glu Asp Leu
Lys Cys Arg Val Leu Glu Leu Pro Tyr Gln Gly Glu 210
215 220Glu Leu Ser Met Val Ile Leu Leu Pro Asp Asp Ile
Glu Asp Glu Ser225 230 235
240Thr Gly Leu Lys Lys Ile Glu Glu Gln Leu Thr Leu Glu Lys Leu His
245 250 255Glu Trp Thr Lys Pro
Glu Asn Leu Asp Phe Ile Glu Val Asn Val Ser 260
265 270Leu Pro Arg Phe Lys Leu Glu Glu Ser Tyr Thr Leu
Asn Ser Asp Leu 275 280 285Ala Arg
Leu Gly Val Gln Asp Leu Phe Asn Ser Ser Lys Ala Asp Leu 290
295 300Ser Gly Met Ser Gly Ala Arg Asp Ile Phe Ile
Ser Lys Ile Val His305 310 315
320Lys Ser Phe Val Glu Val Asn Glu Glu Gly Thr Glu Ala Ala Ala Ala
325 330 335Thr Ala Gly Ile
Ala Thr Phe Cys Met Leu Met Pro Glu Glu Asn Phe 340
345 350Thr Ala Asp His Pro Phe Leu Phe Phe Ile Arg
His Asn Ser Ser Gly 355 360 365Ser
Ile Leu Phe Leu Gly Arg Phe Ser Ser Pro 370
3753379PRTHomo sapiens 3Met Glu Gln Leu Ser Ser Ala Asn Thr Arg Phe Ala
Leu Asp Leu Phe1 5 10
15Leu Ala Leu Ser Glu Asn Asn Pro Ala Gly Asn Ile Phe Ile Ser Pro
20 25 30Phe Ser Ile Ser Ser Ala Met
Ala Met Val Phe Leu Gly Thr Arg Gly 35 40
45Asn Thr Ala Ala Gln Leu Ser Lys Thr Phe His Phe Asn Thr Val
Glu 50 55 60Glu Val His Ser Arg Phe
Gln Ser Leu Asn Ala Asp Ile Asn Lys Arg65 70
75 80Gly Ala Ser Tyr Ile Leu Lys Leu Ala Asn Arg
Leu Tyr Gly Glu Lys 85 90
95Thr Tyr Asn Phe Leu Pro Glu Phe Leu Val Ser Thr Gln Lys Thr Tyr
100 105 110Gly Ala Asp Leu Ala Ser
Val Asp Phe Gln His Ala Ser Glu Asp Ala 115 120
125Arg Lys Thr Ile Asn Gln Trp Val Lys Gly Gln Thr Glu Gly
Lys Ile 130 135 140Pro Glu Leu Leu Ala
Ser Gly Met Val Asp Asn Met Thr Lys Leu Val145 150
155 160Leu Val Asn Ala Ile Tyr Phe Lys Gly Asn
Trp Lys Asp Lys Phe Met 165 170
175Lys Glu Ala Thr Thr Asn Ala Pro Phe Arg Leu Asn Lys Lys Asp Arg
180 185 190Lys Thr Val Lys Met
Met Tyr Gln Lys Lys Lys Phe Ala Tyr Gly Tyr 195
200 205Ile Glu Asp Leu Lys Cys Arg Val Leu Glu Leu Pro
Tyr Gln Gly Glu 210 215 220Glu Leu Ser
Met Val Ile Leu Leu Pro Asp Asp Ile Glu Asp Glu Ser225
230 235 240Thr Gly Leu Lys Lys Ile Glu
Glu Gln Leu Thr Leu Glu Lys Leu His 245
250 255Glu Trp Thr Lys Pro Glu Asn Leu Asp Phe Ile Glu
Val Asn Val Ser 260 265 270Leu
Pro Arg Phe Lys Leu Glu Glu Ser Tyr Thr Leu Asn Ser Asp Leu 275
280 285Ala Arg Leu Gly Val Gln Asp Leu Phe
Asn Ser Ser Lys Ala Asp Leu 290 295
300Ser Gly Met Ser Gly Ala Arg Asp Ile Phe Ile Ser Lys Ile Val His305
310 315 320Lys Ser Phe Val
Glu Val Asn Glu Glu Gly Thr Glu Ala Ala Ala Ala 325
330 335Thr Ala Gly Ile Ala Thr Phe Cys Met Leu
Met Pro Glu Glu Asn Phe 340 345
350Thr Ala Asp His Pro Phe Leu Phe Phe Ile Arg His Asn Ser Ser Gly
355 360 365Ser Ile Leu Phe Leu Gly Arg
Phe Ser Ser Pro 370 375
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