WO1998035235A1 - Dosage de fragments de collagene de type ii - Google Patents

Dosage de fragments de collagene de type ii Download PDF

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Publication number
WO1998035235A1
WO1998035235A1 PCT/GB1998/000304 GB9800304W WO9835235A1 WO 1998035235 A1 WO1998035235 A1 WO 1998035235A1 GB 9800304 W GB9800304 W GB 9800304W WO 9835235 A1 WO9835235 A1 WO 9835235A1
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antibody
epitope
collagen
epitopes
ah12l3
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PCT/GB1998/000304
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English (en)
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Anthony Peter Hollander
Lisa Jane Croucher
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University Of Sheffield
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Priority to EP98902752A priority Critical patent/EP0960339A1/fr
Priority to JP53398298A priority patent/JP2001511253A/ja
Priority to AU59560/98A priority patent/AU5956098A/en
Publication of WO1998035235A1 publication Critical patent/WO1998035235A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • the present invention relates to methods, kits and reagents for assaying for collagen fragments, and to therapeutic, prognostic and diagnostic methods based thereon.
  • the invention relates to type II collagen fragment immunoassays (e.g. Sandwich ELISA), kits and reagents.
  • Type II collagen is the major collagen of hyaline cartilage, accounting for 15-20% of the wet weight of the tissue and 90% of all collagens in cartilage (1).
  • type II collagen molecules become cross-linked with collagen types IX and XI to form heterotypic fibrils, which endow articular cartilage with the tensile strength necessary to withstand the loading forces resulting from day-to-day use of synovial joints (2).
  • Type II collagen is synthesized with a pro-peptide at both termini of the molecule (residues 1-181 and 1242-1487). These are cleaved off as soon as the collagen is released into the extracellular matrix.
  • the mature molecule has a short telopeptide (non-helical peptide) at both termini (residues 182-200 and 1215-1241) and a triple helix about 1000 residues long (residues 201-1214).
  • Inter- and intra-molecular cross- links are located at 4 sites - one in each telopeptide (residues 190 and 1231) and one near each end of the helical region (residues 308 and 1130). Thus, each end of the molecule contains two stable cross-links.
  • Cartilage degradation is a major feature of arthritic diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA)
  • OA osteoarthritis
  • RA rheumatoid arthritis
  • cartilage degradation is presently detected and monitored by X-Rays (which can detect joint space narrowing) or by biopsy (where cartilage removed from arthritic or normal joints at arthroplasty is analysed for the presence of biochemical markers of cartilage breakdown)
  • X-Rays which can detect joint space narrowing
  • biopsy where cartilage removed from arthritic or normal joints at arthroplasty is analysed for the presence of biochemical markers of cartilage breakdown
  • both X-Ray and biopsy procedures are inconvenient, expensive and potentially harmful to the patient Accordingly, there is much interest in alternative methods of monitoring the progress of cartilage breakdown in various arthritic diseases
  • biochemical markers of cartilage breakdown are those a ⁇ smg from a loss of aggrecan (a large, aggregating proteoglycan which contributes to the structural integ ⁇ ty of cartilage) (3-7)
  • Other potential markers are those arising from the degradation of type II collagen, since it is clear that this molecule is damaged in arthritic joints and collagen degradation is generally considered to be an irreversible process (8-11)
  • MMPs matrix metalloprotemases
  • the inhibition ELISA presents two problems with respect to the detection of type II collagen fragments in serum. Firstly, it lacks the sensitivity needed for the detection of fragments that are probably present only at low levels in the systemic circulation. Secondly, it is subject to interference by any protein that can bind to the heat denatured collagen which is used to coat the wells of the ELISA plate.
  • the type II collagen molecule is comprised of regions which differ dramatically in their susceptibility to hydrolysis.
  • the amino-acids falling within the amino terminus cross-link region of the triple helix (residues 201-308) of the type II collagen ⁇ 1 -chain are much less susceptible to hydrolysis by MMPs than the rest of the ⁇ 1 -chain.
  • the cross-linked region at the carboxy-terminus of the triple helix (residues 1130-1214) is less stable, reflected in the lower melting temperature than the amino-terminal end of the molecule (so making it more susceptible to hydrolysis).
  • collagen fragments released from degraded type II cartilage in vivo comprise fragments which are sufficiently large to bear two or more different epitopes, and that the half life of these fragments in vivo is long enough to permit the build up of concentrations high enough to make detection and/or accurate measurement by immunoassay in various body fluids (including serum, urine or synovial fluid) possible.
  • kits comprising a first antibody (e.g. a monoclonal antibody, polyclonal antibody or fragment or derivative thereof) which binds to a first epitope and a second antibody (e.g. a monoclonal antibody, polyclonal antibody or fragment or derivative thereof) which binds to a second epitope, wherein the first and second epitopes are coupled epitopes present on a collagen fragment (for example, a type I or type II collagen fragment) released from degraded cartilage in vivo.
  • the first and second epitopes are coupled epitopes present on C-ll f r ee -
  • C-ll f r ee embraces any of those type II collagen fragments which are released from degraded cartilage, for example by release in vivo as a result of cartilage degradation associated with various diseases or disorders (such as OA and RA).
  • C-ll f ree include collagen fragments which have entered the systemic circulation (hereinafter referred to as C-ll system c ).
  • Such fragments include collagen fragments which may be found in the serum, urine and synovial fluid (the latter abbreviated herein to SF).
  • Coupled epitopes refers to antigenic epitopes which are each present on a single type II collagen fragment or analyte moiety. Coupled epitopes may therefore comprise one or more conformational epitopes, either on a single type II collagen fragment or on a single analyte moiety which comprises a type II collagen fragment complex (e.g. two or more associated collagen fragments which together comprise a single analyte moiety).
  • each coupled epitope is a sequential epitope, so that all coupled epitopes are present on a single type II collagen peptide fragment.
  • Particularly preferred are coupled epitopes comprising or located within an amino acid sequence or a fragment of the N-terminal region of the ⁇ 1 type II collagen chain, for example coupled epitopes comprising or located within an amino acid sequence or a fragment of the N-terminal cross-Hnked region of the triple helix thereof (see Figure 1 ).
  • the first epitope is located within residues 243-263 of the sequence shown in Figure 2
  • the second epitope is located within residues 216-236 of the sequence shown in Figure 3
  • the coupled epitopes may simultaneously bind cognate antibodies, and may advantageously be disposed such that an antibody bound at the first epitope does not stencally hinder (or interfere with) antibody binding at the second epitope
  • the coupled epitopes of the invention may therefore be physically "linked", so that that sequestration or partitioning of one of the epitopes attendant on binding to its cognate antibody effectively also sequesters or partitions the other (linked) epitope (while leaving it available for binding to a second cognate antibody)
  • the coupled epitopes are preferably separated by at least 2 ammo acids, e g by 3,4, 5, 6, 7, 8, 9, 10, 1 1 , 12,13, 14, 15, 16, 17, 18, 19 or 20 ammo acids Preferably, they are separated from each other by at least 3 am o acids (e g about 6 ammo acids) to prevent ste ⁇ c hindrance of simultaneous antibody binding
  • the coupled epitopes are sufficiently separated to permit independent (non-blocking) binding to cognate antibodies, but sufficiently close together to substantially eliminate the possibility of a proteolytic cleavage event uncoupling or separating the epitopes
  • the coupled epitopes are preferably separated by a short ammo acid sequence which is not subject to substantial proteolysis in vivo, for example in a portion of the type II collagen molecule with is relatively stable or which is relatively resistant to proteolysis
  • Particularly preferred is the N terminal portion of the ⁇ 1 type II collagen peptide (e g the N terminal cross-linked region of the triple helix thereof)
  • the immunoassay kit of the invention may be used in therapy, diagnosis, prognosis or candidate drug screening
  • the immunoassay kit of the invention is for use in Sandwich immunoassay (e g for use in Sandwich ELISA)
  • the first antibody may be immobilized on a solid support, and/or the second antibody may be labelled.
  • the immobilized primary antibody is preferably monoclonal (e.g. a mouse monoclonal antibody), while the secondary antibody is preferably polyclonal (e.g. a rabbit polyclonal antibody) and derived from a different species to the capture antibody.
  • the secondary antibody is preferably polyclonal (e.g. a rabbit polyclonal antibody) and derived from a different species to the capture antibody.
  • the anti-lg antibody is preferably labelled with alkaline phosphatase or peroxidase.
  • the secondary antibody may be labelled.
  • Preferred labels include radiolabels and biotin. Where biotin is used, the secondary antibody may be detected by contacting the secondary antibody with enzyme-labelled avidin.
  • the kit comprises an enzyme-labelled reactant
  • the kit preferably further comprises a substrate to react with the enzyme label.
  • the kit may further comprise standard heat denatured type II collagen or a standard synthetic peptide carrying C-M ⁇ e coupled epitopes (e.g. AH8 and AH12).
  • Such synthetic peptides may comprise a protease-resistant spacer region between the coupled epitopes.
  • Such synthetic peptides may be synthesised, for example, by solid phase peptide synthesis or by expression of synthetic gene(s) in a recombinant host cell.
  • AH8, AH9 and AH 12 are used to refer to particular peptide epitopes or peptides per se (as sense dictates and as described in more detail infra).
  • AH8L1 , AH9L2 and AH12L3 refer to the rabbit antisera to the AH8, AH9 and AH 12 peptides, respectively.
  • AH8MAb refers to a monoclonal antibody to peptide AH8.
  • anti-AH8, anti-AH9 and anti-AH12 refer to antibodies raised against (or (specifically) reactive with) the peptides AH8, AH9 and AH 12, respectively.
  • the first antibody is AH8MAb or AH8L1 and the second antibody is AH12L3.
  • the first antibody may be AH12L3 and the second antibody AH ⁇ MAb or AH8L1.
  • the first antibody may also be an antibody which competitively inhibits binding of AH ⁇ MAb or AH8L1 to cognate epitope
  • the second antibody may be an antibody which competitively inhibits binding of AH12L3 to cognate epitope
  • the first antibody may be an antibody which competitively inhibits binding of AH12L3 to cognate epitope
  • the second antibody may be an antibody which competitively inhibits binding of AH8MAb or AH8L1 to cognate epitope.
  • the first antibody may be an antibody having the same (or essentially the same) epitope specificity as antibody AH ⁇ MAb or AH8L1
  • the second antibody may be an antibody having the same (or essentially the same) epitope specificity as antibody AH12L3.
  • the first antibody may be an antibody having the same (or essentially the same) epitope specificity as antibody AH12L3
  • the second antibody may be an antibody having the same (or essentially the same) epitope specificity as antibody AH ⁇ MAb or AH8L1.
  • Binding agents other than antibodies having functionally similar properties to the antibodies of the invention may also be used according to the invention.
  • the first and/or second antibody may be a binding agent derived from, comprising or consisting essentially of the variable region of an antibody having the required specificity (for example antibody AH8MAb, AH8L1 or AH12L3).
  • Binding agents for use in the invention therefore include various antibody fragments, such as Fab, Fab' or F(ab') 2 fragments, or synthetic proteins and peptides modelled on sequences variable regions having the desired specificity.
  • the invention relates to an antibody (e.g. a monoclonal antibody, polyclonal antibody, or fragment or derivative thereof) per se which binds to a coupled epitope of C-li f r ee -
  • the antibodies of the invention may be monoclonal or polyclonal, and are preferably labelled. Preferred labels include biotin and radioactive labels.
  • the antibodies of the invention may also be immobilized (e.g. on a solid support, such as the well of a microtitre plate or surface of a probe or dipstick).
  • heteroa ⁇ tibodies comprising a first binding moiety which binds to a first epitope and a second binding moiety which binds to a second epitope, wherein the first and second epitopes are C-ll free coupled epitopes.
  • Such heteroantibodies find application in various immunoprecipitation assays, since they form readily detectable high molecular weight complexes in the presence of coupled C-ll f r ee epitopes.
  • a biological sample e.g. in vitro or ex vivo
  • the methods of the invention find application in various in vitro or ex vivo methods of therapy, diagnosis or prognosis comprising the step of assaying a biological sample for C-ll fre e.
  • the invention also contemplates a process for measuring the therapeutic activity of an agent comprising the steps of: (a) providing a sample derived from a subject treated with the agent; and (b) assaying the sample for C- ⁇ free using the kit, antibody/binding agent or the method of the invention, wherein the amount of C-ll f r ee in the sample assayed in step (b) is used as an index of therapeutic activity.
  • the invention provides a process for producing a therapeutic agent comprising the step of screening a library of candidate therapeutic agents for therapeutic activity using the therapeutic activity measuring process of the invention described above.
  • the invention contemplates a process for producing a therapeutic agent comprising the steps of: (a) selecting a chemical formula on the basis of one or more structural features of a therapeutic agent produced by the process for producing a therapeutic agent of the invention; and then (b) producing (e.g. by synthesis or isolation) a therapeutic agent having the chemical formula selected in step (a). Also covered are therapeutics obtainable by (or obtained by) this process. These preferably include anti-arthritic drugs and MMP inhibitors.
  • the invention contemplates a cell or cell line which expresses the antibody or binding agent of the invention.
  • the cell or cell line may be prokaryotic or eukaryotic (e.g. mammalian). Preferred are hybridomas expressing monoclonal antibodies according to the invention.
  • a process for producing an antibody which binds to a coupled epitope of C-ll free comprising the steps of: (a) degrading type II collagen in vitro to produce a plurality of fragments; (b) fractionating the fragments on the basis of size; (c) identifying relatively large fragments; (d) screening the relatively large fragments for the presence of multiple epitopes; (e) selecting a fragment which bears two or more epitopes; and (f) raising antibodies against the fragment selected in step (e).
  • antibodies which bind to a coupled epitope of C- ll f ree obtainable (or obtained by) the process described above.
  • the invention also covers isolated type II collagen fragments bearing two or more epitopes for use in step (f) of the processes of the invention, as well as processes for producing an antibody or C-ll f re e binding agent comprising raising antibodies against the isolated fragments of the invention.
  • the invention finds application in various assays, including diagnostic, prognostic and drug screening assays.
  • the invention may also be used as part of a therapeutic regimen, as described below.
  • the invention may be useful in various forms of therapy, prognosis and diagnosis.
  • the sandwich ELISAs of the invention are sensitive enough to detect type II collagen fragments in urine, serum or SFs from patients.
  • the samples can be easily obtained without risk to the patient, and the assay is simple and inexpensive.
  • routine screening of a population for arthritic or other diseases in which cartilage breakdown occurs is possible using the methods and reagents of the invention
  • the invention can also be used to determine the degree of on-going cartilage collagen degradation in patients with different types of OA or RA
  • the assays may therefore also find utility as the basis of prognostic tests, for example to predict which patients will go on to develop severe cartilage lesions
  • the invention provides a means of determining the in vivo efficacy of anti- arth ⁇ tic drugs, such as MMP inhibitors
  • anti- arth ⁇ tic drugs such as MMP inhibitors
  • a number of such drugs are about to be tested in clinical t ⁇ als (3, 26), but prior to the invention there has been no way of following type II collagen degradation using a urine, serum or SF marker, to determine if the patients are protected by treatment
  • the invention may therefore find application as an adjunct to va ⁇ ous forms of therapy or as a means for determining the optimum dosage and/or frequency of administration of therapeutic drugs
  • assays of the invention include the diagnosis of growth disorders and the testing of the efficacy or monitoring the progress of growth hormone treatment
  • long bone growth depends on the process of endochondral ossification, in which growth plate cartilage is degraded and replaced with bone (1)
  • Serum levels of type II collagen fragments will therefore be higher in normal growing children than in adults, reflecting this process
  • the serum levels of these fragments are likely to be lower than is normal for children of any given age
  • the invention may be used to diagnose such disorders, as an adjunct to drug treatment to establish dose-response relationships and also to monitor the progress of the disorder and its treatment
  • Figure 1 shows schematically the different molecular zones or domains in the type II collagen ⁇ -chain and the location of the eprtopes used to generate antibodies 1 epitope AH12 2 epitope AH8
  • Figure 2 shows the primary structure (amino acid sequence) of the human type II collagen ⁇ -chain.
  • Figure 3 shows schematically the Inhibition ELISA assay format
  • type II collagen fragments in solution phase bind to primary antibody, inhibiting it from binding to the solid phase collagen.
  • Panel B Sandwich ELISA
  • type II collagen fragments in solution phase bind to the primary antibody via the first epitope on the collagen fragments.
  • biotinylated secondary anti-type II collagen antibody binds to second epitope on collagen fragments.
  • Figure 4 shows the characterization of antiserum AH8L1.
  • Antiserum AH6L1 was tested in ELISA assays against peptide AH8 (Panel A) and HDC (Panel B). The results are shown for specific antiserum (circles) compared to the pre-immune serum (diamonds) for both antigens.
  • Figure 5 shows the characterization of antiserum AH9L2.
  • Antiserum AH9L2 was tested in ELISA assays against peptide AH9 (Panel A) and HDC (Panel B). The results are shown for specific antiserum (circles) compared to the pre-immune serum (diamonds) for both antigens.
  • Figure 6 shows the characterization of antiserum AH12L3. Antiserum AH12L3 was tested in ELISA assays against peptide AH 12 (Panel A) and HDC (Panel B). The results are shown for specific antiserum (circles) compared to the pre-immune serum (diamonds) for both antigens.
  • Figure 7 shows the cross-reactivity of anti-peptide anti-sera with different collagens.
  • lane 1 is human type 1 collagen
  • lane 2 is bovine type I collagen
  • lane 3 is human type II collagen
  • lane 4 is bovine type II collagen
  • lane 5 is human type III collagen
  • lane 6 is bovine type III collagen
  • lane 7 is human type IV collagen.
  • Figure 8 shows Western immunoblots of bovine chondrocyte lysates.
  • the lysates were prepared with (lane 1) or without (lane 2) the addition of bovine type II collagen.
  • Panel A is an amido black stain of total protein in the lysates after transfer to nitrocellulose.
  • Panels B, C and D are Western immunoblots using antibodies AH8L1 , AH9L2 and AH12L3 respectively. The migration position of the type II collagen ⁇ -chain is shown.
  • Figure 9 shows Western immunoblots of week 3 medium from bovine nasal cartilage cultures.
  • Medium from IL-1 -stimulated cartilage cultures was separated by SDS-PAGE on 15% gels and electroblotted to a nitrocellulose membrane.
  • Panel A shows an amido black stain of total protein in the lysates after transfer to nitrocellulose.
  • Panels B, C, D and E show Western immunoblots with antibodies Pa-3/4, AH8L1 , AH9L2 and AH12L3, respectively. The migration position of the two most clearly defined collagen fragments are indicated, as well as the molecular weight standards.
  • Figure 10 shows the location of the amino-terminus of fragment TII-F1 within the type II collagen triple helix.
  • Figure 11 shows dual binding of AH8L1 and AH12L3 to HDC.
  • Purified, biotinylated antibody AH ⁇ L1 was bound to HDC on its own (circles in both panels) and following preincubation of the HDC with antiserum AH12L3 at a 1 :100 dilution (diamonds in Panel A) or with purified antibody AH 12L3 at a 1 :64 dilution (diamonds in Panel B). Binding of AH8L1 was detected using the avidin-peroxidase conjugate method described infra.
  • Figure 12 shows a comparison of type II collagen assays. HDC was assayed at a range of standard concentrations either using the Sandwich ELISA of the invention (Panel A) or the known CB11 B inhibition ELISA (Panel B).
  • Figure 13 shows immunohistochemical staining of human OA femoral head cartilage from a human subject. Staining was with mouse monoclonal antibody Col2- 3/4m (A and D), rabbit polyclonal antibody AH12L3 (B and E) and rabbit polyclonal antibody AH9L2 (C and F). Negative controls (non-immune IgG) are shown separately for mouse (G) and rabbit (H).
  • Figure 14 shows immunohistochemical staining of human normal femoral head cartilage from a human subject. Staining was with mouse monoclonal antibody Col2- 3/4m (A), rabbit polyclonal antibody AH12L3 (B) and rabbit polyclonal antibody AH9L2 (C). Negative controls (non-immune IgG) are shown separately for mouse (D) and rabbit (E).
  • Figure 15 shows dual binding of antibodies AH ⁇ MAb and AH 12 to HDC.
  • Purified antibody AH12 was bound to HDC either on its own (squares) or following preincubation of the HDC with AH ⁇ MAb (circles) and was detected using an alkaline phosphatase labeled goat anti-rabbit Ig second antibody.
  • Figure 16 shows the AH ⁇ MAb/AH12L3 sandwich ELISA standard curve. Results are shown as the mean +/- SEM for 11 separate experiments, using type II collagen purified from bovine nasal septum cartilage.
  • Figure 17 shows specific detection of denatured type II collagen by the AH ⁇ MAb/AH12L3 sandwich ELISA.
  • Native bovine type II collagen was prepared as described in Example 5 and then a portion was denatured. The samples were then assayed at a series of dilutions.
  • Figure 20 shows the standard synthetic peptide for use in the AH3MAb/AH12L3 sandwich ELISA.
  • Amino acid sequences from the amino- and carboxy-terminal cross-linked regions of the ⁇ 1(ll) chain were subjected to hydrophobicity analysis and suitable immunogenic peptides were selected using the criteria previously described (10).
  • Peptides AH ⁇ , AH9 and AH 12 were each synthesized with an amino-terminal cysteine for coupling to carrier protein and a carboxy terminal tyrosine for possible iodination.
  • AH ⁇ and AH 12 are located in the amino-terminal cross-linked region of the triple helix and AH9 is in the carboxy-terminal cross-linked region, as shown in Figure 1.
  • the peptides were synthesized by Dr. Arthur Moir in the Kreb's Institute, University of Sheffield and their sequences are:
  • Each of these peptides was conjugated to the carrier protein, Keyhole Limpet Haemocyanin (Calbiochem, Nottingham, UK), through its amino-terminal cysteine residue, using the coupling reagent bromoacetic acid-N-hydroxysuccinimide ester (Sigma). The method of conjugation was exactly as previously described for peptide coupling to ovalbumin (10).
  • Heat denatured type II collagen was prepared by dissolving bovine type II collagen (Sigma) in 0 1M carbonate buffer (0.1M NaHCO 3 , 0 1M Na 2 CO 3 , pH 9 2) and heating at 60°C for twenty minutes lmmulon-2 ELISA plates (Dynatech Labs, Guernsey, UK) were coated with 2 ⁇ g per well of HDC by passive adsorption for 72 hours at 4°C The plates were washed three times with PBS containing 0 1 % v/v Tween-20 (Sigma, PBS-Tween) and then blocked by incubation with 1% w/v bovine serum albumin (BSA, Sigma) in PBS, for thirty minutes at room temperature The plates were washed once with PBS-Tween and Rabbit antisera were added to wells of the ELISA plates at a range of dilutions
  • the membranes were incubated for thirty minutes at room temperature with the alkaline phosphatase-conjugated goat anti-mouse (for Pa-3/4) or anti-rabbit (for AH ⁇ , 9 and 12) second antibody, diluted 1 :1000 with PBS-3% BSA.
  • the membranes were washed three times with PBS-Tween and once with distilled water.
  • Alkaline phosphatase substrate solution was prepared from a kit (Bio-Rad) employing 5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium. It was added to membranes and incubated at room temperature until optimal colour had developed. The reaction was stopped by rinsing with distilled water.
  • Freshly dissected cartilage from bovine nasal septum was sectioned into slices of 5cm x 1.5cm x 2mm with a scalpel. The slices were washed once with sterile PBS for twenty minutes. Discs of cartilage (40mg average wet weight) were prepared using a sterile stainless steel punch. The outer parts of the cartilage slices were not used to avoid contamination with other tissues.
  • the nasal and articular cartilage explants were cultured in serum free Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies Ltd., Renfrew Road, Paisley, UK), containing glutamine (2mM), penicillin G (2000 U/ml) and streptomycin (0.1 mg/ml) and 4-(2-hydoxyethyl)-1-piperazineethanesulfonicacid (HEPES, 10mM) (all from Gibco) for four weeks in 4 ⁇ -well tissue culture plates (Costar, High Wycombe, UK) at 37°C in a humidified atmosphere of 5% CO 2 /95% air. Each well contained two discs in 400 ⁇ l of medium.
  • DMEM serum free Dulbecco's modified Eagle's medium
  • HEPMS 4-(2-hydoxyethyl)-1-piperazineethanesulfonicacid
  • the discs were treated with recombinant human IL-1 ⁇ was a kind gift from Dr. Michael B. Widmer, Immunex Corp., Seattle, USA.
  • medium in each well was replenished, including fresh IL-1 ⁇ .
  • Antibodies were purified and biotinylated using a blotting membrane as an antigen support, according to the newly described method of Rucklidge et al (2 ⁇ ).
  • AH ⁇ L1 and AH12L3 antibodies were both affinity purified from immune rabbit sera using PVDF membranes (Bio-Rad) coated with HDC.
  • AH ⁇ L1 antibody was biotinylated while still bound to the membrane by incubating with biotin (Biotinamidocaproate N- hydroxysuccinimide ester, Sigma) at 75 ⁇ g/ml in 0.1 M carbonate buffer for thirty minutes, before stripping from the membrane.
  • biotin Biotinamidocaproate N- hydroxysuccinimide ester, Sigma
  • Capture antibody (affinity purified AH12L3) at a dilution of 1 :8 in 0.1 M carbonate buffer was allowed to passively bind to an Immulon 2 plate at 37°C for one hour. All wells were then blocked for one hour at 37°C with carbonate buffer containing 0.2% BSA, then washed three times with PBS/Tween. HDC was diluted in PBS, added to appropriate wells and incubated for ninety minutes at 37°C.
  • detecting antibody biotinylated AH8L1
  • PBS/Tween/0.1 % BSA a further three washes in PBS/Tween
  • detecting antibody biotinylated AH8L1
  • Peroxidase substrate o-phenolenediamine dihydrochloride, Sigma
  • a concentration of 0.5mg/ml in 0.05M citric acid with 0.1M Na 2 HPO 4 and 10 ⁇ l H 2 O 2 was added to all wells and colour developed for 20-30 minutes before stopping with 10M H 2 SO .
  • Absorbences were read at 490nm.
  • Antibodies AH8L1 , AH9L2 and AH12L3 were each tested in western immunoblotting for reactivity with different types of heat denatured collagen. Each of them recognised only type II collagen and not collagen types I, III or IV ( Figure 7), however AH9L2 was less effective at detecting the type II collagen by this technique than AH6L1 or AH12L3.
  • each antibody was tested in a Western immunoblot against the many proteins present in chondrocyte lysates. None of these proteins were detected by any of the antibodies whereas they each detected type II collagen which had been added to the lysates ( Figure ⁇ ).
  • a preliminary Sandwich ELISA was set up by coating unlabelled antibody AH12L3 onto the ELISA plates, incubating with HDC and then detecting with biotinylated antibody AH ⁇ L1 followed by avidin-peroxidase conjugate. HDC was detected by this assay and the lower detection limit was about 0.05 ⁇ g/ml type II collagen ( Figure 12A).
  • Cartilage pieces were embedded in OCT embedding medium (B.D.H. Laboratory Supplies, Poole, England) and sections 7 ⁇ m thick cut and attached to slides pre- coated with 3-aminopropyltriethoxysilane (I. ON. Biomedicals Ltd., Thame, England) as previously described (1 1), to ensure maximum adherence of the section for uniform staining. Sections were either stained immediately or stored at -20°C. Aldeyde groups were blocked with normal goat serum, diluted 1 :10, and permeability of the extracellular matrix was maximised with Chondroiti ⁇ ase ABC lyase (I.C.N.) containing proteinase inhibitors.
  • OCT embedding medium B.D.H. Laboratory Supplies, Poole, England
  • 3-aminopropyltriethoxysilane I. ON. Biomedicals Ltd., Thame, England
  • Sections were probed with Col2-3/4m, AH9L2 or AH12L3. Optimum dilutions were determined by matching the IgG concentration of the antibody with that of a suitable control antibody.
  • Col2-3/4m which is an lgG1
  • a mouse monoclonal was used of the same isotype but raised to an irrelevant epitope.
  • IgG purified from the rabbit anti-peptide antisera AH9L2 and AH12L3 a control IgG from non-immune rabbits (Vector Laboratories) was used. These control antibodies were diluted to the highest concentration which produced negligible background staining of cartilage specimens. A biotin-avidin detection system was used.
  • Sections were incubated with biotin-labelled secondary antibody, goat-anti-rabbit or mouse lg(H+L) (Southern Biotechnology Associates), diluted 1 :100. Bound antibody was detected with the ABC Elite biotin-streptavidin kit (Vector Laboratories) (avidin-biotin-peroxidase) according to the manufacturer's directions and the peroxidase reaction was developed using 3,3'-diaminobenzidine (DAB) (Vector Laboratories), with the addition of nickel to form a grey-black stain. Sections were dehydrated through graded alcohols before mounting permanently with DPX (B.D.H.).
  • biotin-labelled secondary antibody goat-anti-rabbit or mouse lg(H+L) (Southern Biotechnology Associates)
  • Bound antibody was detected with the ABC Elite biotin-streptavidin kit (Vector Laboratories) (avidin-biotin-peroxidase) according to the manufacturer's directions and
  • Figure 13 shows a typical example of OA femoral head cartilage. There were differences in the intensity as well as the pattern of staining between the 3 antibodies. The results with mouse monoclonal Col2-3/4m were essentially as described previously (11). Staining was seen throughout the depth of the OA cartilage, with a thin band of intense staining at the articular surface and moderate staining extending into the mid-zone (Figure 13A). Deep zone chondrocytes exhibited strong pericellular staining ( Figure 13D).
  • Figure 14 shows a typical example of non-arthritic femoral head cartilage.
  • the pattern of staining was similar to that previously described (1 1 ).
  • the interterritorial staining seen with Col2-3/4m and AH12L3 was of a low intensity and restricted mainly to a band of staining at the articular surface ( Figure 14, A and B)
  • the extent and intensity of staining around chondrocytes was also much reduced There was virtually a complete absence of staining with AH9L2, in most cases at the same level as the non-immune control ( Figure 14C)
  • a monoclonal antibody was raised to the AH8 epitope for use with AH12L3 in a sandwich ELISA BALB/c mice were immunized subcutaneously 5 times with KLH- conjugated AH8L1 , 10O ⁇ g of peptide per injection The first injection was emulsified with Complete Freund's Adjuvant, subseguent injections in Incomplete Freund's Adjuvant Splenocytes from all of the mice were pooled, fused to SP2/0 myeloma cells and a monoclonal antibody was prepared using conventional technology A positive clone was chosen on the basis of the ability of its secreted antibody to react with AH ⁇ and HDC by ELISA (as described above, using goat-anti-mouse Ig-alkaline phosphatase (Southern Biotechnology) as the second antibody) The chosen clone was expanded in a spinner flask, at the Sheffield Hyb ⁇ doma Unit, Sheffield University The resulting crude antibody preparation was applied to
  • AH ⁇ MAb was found to be specific for the AH ⁇ peptide and HDC by ELISA, showing minimal cross-reactivity with AH 12 and AH9 peptide. As with the AH ⁇ L1 polyclonal antibody, it was also found not to bind to type I, III or IV collagens, and was positive for the 3/4 piece of collagenase-cleaved type II collagen only, shown by Western Blotting. AH ⁇ MAb also detected the same bands in week 3 medium from IL-1 ⁇ stimulated BNC as polyclonals AH ⁇ L1 and AH12L3 (data not shown).
  • Serum samples diluted in PBS as required, were added in duplicate to test wells at 50 ⁇ l/well. The plate was then incubated for 90 minutes at 37°C, before washing 3 times in PBS-Tween. Secondary antibody, AH12L3, was diluted to 1 :500 in PBS containing 0.1% Tween 20 and 3% BSA, and 50 ⁇ l added to all wells. After a further incubation at 37°C for 90 minutes, the plate was washed as above and third antibody, peroxidase conjugated goat anti-rabbit Ig (Sigma Biosciences) was added to all wells at 1 :1000 in PBS-Tween-BSA at 50 ⁇ l/well, and incubated at 37°C for 90 minutes.
  • Epitopes AH ⁇ and AH 12 are located close to each other and so it was important to demonstrate that monoclonal A ⁇ MAb and polyclonal AH12L3 could both bind to denatured type II collagen at the same time as each other.
  • Figure 15 demonstrates the ability of AH12L3 to bind to its epitope on HDC in the presence of previously-bound AH ⁇ MAb. It can be seen that there is no diminution of binding of AH12L3, as this curve is almost identical to that produced when AH12L3 is bound to HDC alone. This indicates that although the AH ⁇ and AH 12 epitopes are in close proximity to each other, both antibodies are able to bind to their respective epitopes, without interference from each other. This allows detection of fragments of type II collagen bearing these epitopes in serum or synovial fluid when assayed in the sandwich ELISA.
  • bovine type II collagen can be used as a standard for the assay, there may be batch to batch variation in this standard.
  • a 50-residue peptide which contains both epitopes AH ⁇ and AH 12 was synthesised.
  • the sequence is shown in Figure 20. This standard may be used in assays designed to monitor disease-related changes in serum-levels of type II collagen fragments.

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Abstract

L'invention a pour objet des procédés, des trousses et des réactifs pour doser des fragments de collagène, et des procédés thérapeutiques, pronostiques et diagnostiques utilisant ces derniers.
PCT/GB1998/000304 1997-02-06 1998-01-30 Dosage de fragments de collagene de type ii WO1998035235A1 (fr)

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EP98902752A EP0960339A1 (fr) 1997-02-06 1998-01-30 Dosage de fragments de collagene de type ii
JP53398298A JP2001511253A (ja) 1997-02-06 1998-01-30 ▲ｉｉ▼型コラーゲンフラグメントについてのアッセイ
AU59560/98A AU5956098A (en) 1997-02-06 1998-01-30 Assay for collagen type ii fragments

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GBGB9702252.9A GB9702252D0 (en) 1997-02-06 1997-02-06 Collagen assay
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0921395A2 (fr) * 1997-11-13 1999-06-09 Pfizer Products Inc. Méthode pour déterminer des fragments de la protein dans les milieux biologiques
EP1130401A2 (fr) * 2000-02-15 2001-09-05 Pfizer Products Inc. Essais pour la mesure des fragments de collagène type II dans l'urine
EP1149111A1 (fr) * 1999-01-06 2001-10-31 University Of Southern California Technique et composition pour l'inhibition de l'angiogenese
EP1215496A1 (fr) * 1999-09-13 2002-06-19 Nissui Pharmaceutical Co., Ltd. Procede et kit de detection ou de dosage d'une substance
FR2865034A1 (fr) * 2004-01-14 2005-07-15 Synarc Procede d'evaluation de la degradation du tissu cartilagineux d'un individu
WO2005106495A1 (fr) * 2004-05-04 2005-11-10 Pharmacia & Upjohn Company Llc Biomarqueurs de procollagene ii et leurs methodes
US7195883B2 (en) * 1996-12-09 2007-03-27 Osteometer Biotech A/S Sandwich assays for collagen fragments
US7365167B2 (en) 2001-11-26 2008-04-29 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7390885B2 (en) 2001-11-26 2008-06-24 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7488792B2 (en) 2002-08-28 2009-02-10 Burnham Institute For Medical Research Collagen-binding molecules that selectively home to tumor vasculature and methods of using same
US7566449B2 (en) 1999-07-13 2009-07-28 University Of Southern California Method and composition for inhibition of angiogenesis using antagonists based on MMP-9 and β1 integrins
US9086418B2 (en) 2005-11-01 2015-07-21 Abbvie Biotechnology Ltd. Methods and compositions for diagnosing ankylosing spondylitis using biomarkers

Citations (2)

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WO1994014070A1 (fr) * 1992-12-04 1994-06-23 Shriners Hospitals For Crippled Children Immunodosage pour la mesure du clivage collagenique dans le cartilage
WO1994018563A1 (fr) * 1993-02-12 1994-08-18 Rhode Island Hospital Detection de collagene de type ii et de ses peptides

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WO1994014070A1 (fr) * 1992-12-04 1994-06-23 Shriners Hospitals For Crippled Children Immunodosage pour la mesure du clivage collagenique dans le cartilage
WO1994018563A1 (fr) * 1993-02-12 1994-08-18 Rhode Island Hospital Detection de collagene de type ii et de ses peptides

Non-Patent Citations (1)

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Title
K. MORGAN ET AL.: "Identification of an immunodominant B-cell epitope in bovine type II collagen and the production of antibodies to type II collagen by immunization with a synthetic peptide representing this epitope.", IMMUNOLOGY, vol. 77, no. 4, December 1992 (1992-12-01), OXFORD, GB, pages 609 - 616, XP002069246 *

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7195883B2 (en) * 1996-12-09 2007-03-27 Osteometer Biotech A/S Sandwich assays for collagen fragments
EP0921395A3 (fr) * 1997-11-13 1999-09-01 Pfizer Products Inc. Méthode pour déterminer des fragments de la protein dans les milieux biologiques
EP0921395A2 (fr) * 1997-11-13 1999-06-09 Pfizer Products Inc. Méthode pour déterminer des fragments de la protein dans les milieux biologiques
US6642007B1 (en) 1998-11-02 2003-11-04 Pfizer Inc. Assays for measurement of type II collagen fragments in urine
US7345151B2 (en) 1999-01-06 2008-03-18 University Of Southern California Antagonists specific for denatured collagen and methods of using same
US7122635B2 (en) 1999-01-06 2006-10-17 University Of Southern California Method and composition for angiogenesis inhibition
JP2002539076A (ja) * 1999-01-06 2002-11-19 ユニバーシティ・オブ・サザン・カリフォルニア 新脈管形成抑制のための方法および組成物
US7588760B2 (en) 1999-01-06 2009-09-15 University Of Southern California Antagonists specific for denatured collagen and methods of using same
EP1149111A4 (fr) * 1999-01-06 2004-08-11 Univ Southern California Technique et composition pour l'inhibition de l'angiogenese
US8025883B2 (en) 1999-01-06 2011-09-27 University Of Southern California Antagonists and methods for inhibiting angiogenesis
JP2011084566A (ja) * 1999-01-06 2011-04-28 Univ Of Southern California 新脈管形成抑制のための方法および組成物
EP1149111A1 (fr) * 1999-01-06 2001-10-31 University Of Southern California Technique et composition pour l'inhibition de l'angiogenese
US7566449B2 (en) 1999-07-13 2009-07-28 University Of Southern California Method and composition for inhibition of angiogenesis using antagonists based on MMP-9 and β1 integrins
EP1215496A4 (fr) * 1999-09-13 2005-06-08 Nissui Pharm Co Ltd Procede et kit de detection ou de dosage d'une substance
EP1215496A1 (fr) * 1999-09-13 2002-06-19 Nissui Pharmaceutical Co., Ltd. Procede et kit de detection ou de dosage d'une substance
EP1130401A2 (fr) * 2000-02-15 2001-09-05 Pfizer Products Inc. Essais pour la mesure des fragments de collagène type II dans l'urine
EP1130401A3 (fr) * 2000-02-15 2002-07-17 Pfizer Products Inc. Essais pour la mesure des fragments de collagène type II dans l'urine
US7763247B2 (en) 2001-11-26 2010-07-27 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7763248B2 (en) 2001-11-26 2010-07-27 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7365167B2 (en) 2001-11-26 2008-04-29 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7390885B2 (en) 2001-11-26 2008-06-24 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7566770B2 (en) 2001-11-26 2009-07-28 Cell-Matrix, Inc. Humanized collagen antibodies and related methods
US7488792B2 (en) 2002-08-28 2009-02-10 Burnham Institute For Medical Research Collagen-binding molecules that selectively home to tumor vasculature and methods of using same
EP1555531A1 (fr) * 2004-01-14 2005-07-20 Synarc Procédé d'évaluation de la dégradation du tissu cartilagineux d'un individu
FR2865034A1 (fr) * 2004-01-14 2005-07-15 Synarc Procede d'evaluation de la degradation du tissu cartilagineux d'un individu
WO2005106495A1 (fr) * 2004-05-04 2005-11-10 Pharmacia & Upjohn Company Llc Biomarqueurs de procollagene ii et leurs methodes
US9086418B2 (en) 2005-11-01 2015-07-21 Abbvie Biotechnology Ltd. Methods and compositions for diagnosing ankylosing spondylitis using biomarkers

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