JP2001511253A - Assay for type II collagen fragment - Google Patents

Abstract

  (57) [Summary] Methods, kits, and reagents for assaying for collagen fragments, and therapeutic, prognostic, and diagnostic methods based thereon are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION               Assay for type II collagen fragmentsField of the invention   The present invention relates to methods, kits for assaying for collagen fragments. , And reagents, and therapeutic, prognostic, and diagnostic methods based thereon. Special In addition, the present invention provides a type II collagen fragment immunoassay (eg, a sandwich ELISA, kits, and reagents.Introduction   Type II collagen is 15-20% of the wet weight of tissue and all collagen in cartilage It is the major collagen of hyaline cartilage, which accounts for 90% of the total amount of collagen (1). Extracellular matrix In type II collagen molecules cross-link with type IX and type XI collagen Forming a brill, which is the loading for the daily use of the synovial joint. Attach articular cartilage with the tension necessary to resist ce) (2).   There are many important structural features of the collagen molecule (23), and these A schematic diagram of the gen molecule (FIG. 1). The primary structure of human type II collagen α1 chain is The numbers of all amino acid residues shown in FIG. 2 and cited herein are Means the amino acid sequence shown in FIG.   I with propeptides (residues 1-181 and 1242-1487) at both ends of the molecule Type I collagen is synthesized. They release collagen into the extracellular matrix. Disconnects as soon as it is served. The mature molecule is located at both ends (residues 182-200 and 1215 ~ 1241) a short telopeptide (non-helical peptide) and a triple of about 1000 residues long It has a helix (residues 201 to 1214). Inter- and intra-molecular cross-links consist of four sites- One for each telopeptide (residue 190 and residue 1231) and one for the helical region Located near each end of the region (residues 308 and 1130). Therefore, the molecular Each end contains two stable bridges.   Cartilage degradation can occur in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). It is a major feature of the case. Monitor the progression of cartilage loss in such diseases Is currently of great interest in disease management (including prognosis, diagnosis, and treatment) Provide useful information. At the end of this, cartilage degradation is Can detect the narrowing of the cavity) or by biopsy (arthroplasty) In surgery, cartilage removed from arthritis or normal joints is a biochemical marker of cartilage breakdown. Analyzed for the presence of a car), detected and monitored. However, X-ray Both biopsy and biopsy procedures are inconvenient, expensive, and possibly patient May be harmful. Therefore, the progress of cartilage breakdown in various joint diseases can be monitored. An interesting alternative is very interesting.   Among the known biochemical markers of cartilage destruction are aggrecan (the structural integrity of cartilage). (3-7) may result from the loss of large aggregated proteoglycans that contribute to ). Other potential markers are that this molecule is damaged in arthritic joints and Type II collagen, since genolysis is generally considered to be an irreversible process. It arises from the decomposition of (8-11).   At present, collagen degradation in cartilage depends on matrix metalloproteinase (MMP). There is good evidence that it is mediated by one class of enzymes called. In particular , Collagenases (MMP-1 and MMP-13) break the triple helix of type II collagen Is considered to be responsible for the breakdown of this molecule and subsequent cartilage degradation by gelatinase. This results in removal from Trix (12-14). In synovial joints, extracellular matrix Quench fragments are released from decomposing cartilage into synovial fluid (SF), which Lubricate the surface.   Therefore, these fragments are found at high concentrations in SF from arthritis patients. (15-19). They pass from the SF through the lymphatic system to the systemic circulation. Serum levels of cartilage matrix protein fragments are usually higher than in SF. Levels of specific marker proteins in serum in OA and RA It is further possible to detect a change.   There are many assays available to measure aggrecan degradation products (17, 20-22) Currently, type II collagen in the systemic circulation (eg, in serum or urine) There is no suitable assay for genogen fragments.   Two major questions related to measuring systemic type II collagen fragments There is a title. First, type II collagen turnover in articular cartilage is not normally Very low, and therefore of type II collagen fragments in serum or urine Reliable levels are very low and therefore difficult to detect and measure accurately. is there. Although there is an increase in the rate of type II collagen turnover in OA cartilage, No change is large enough to significantly facilitate detection. Therefore, high sensitivity New assays are required. The second problem is circulating type II collagen fragments Is small due to extensive cleavage by the gelatin-melting enzyme . This is a polyclonal strategy for multiple epitopes on denatured collagen. Means that the antibody cannot be used to initiate a "sandwich" ELISA Because this assay format allows two different epitopes to be Because it must be present in a single acting collagen fragment (Figure 3B checking).   Therefore, detecting a single collagen epitope by using an inhibition ELISA It has been proposed (see Figure 3A). For this, the molecule is denatured Detects exposed type II collagen epitopes, but does not Assays have developed an assay that is hidden by conformational constraints (10) . Here, the monoclonal antibody Pa-3 / 4 (also known as Col2-3 / 4m) was 1B, which recognizes denatured type II collagen but recognizes other fibrous collagen Not recognize the gene. Inhibition ELISA based on this antibody Detecting type II collagen fragments with osteoarthritis and non-arthritis cartilage Shows increased levels of denatured type II collagen extracted from OA articular cartilage Was reported.   However, at present, this assay is actually a type II collagen in serum and SF. It has been found that it is not useful for detecting fragments. Collage in serum The large value measured for non-binding fragments indicates that the collagen-binding protein (eg, For example, fibronectin) is the result of interference in an inhibition Resulting in positive data. In fact, at present, interfering proteins Removed by cleavage with tainase K, an enzyme that does not damage the CB11B epitope Found that serum type II collagen could not be detected by the assay ing. Factors that further contribute to this negative result are the standard The curve appears to be steep and insensitive: lower detection limit The world is the area of 50 μg / ml type II collagen.   Therefore, the inhibition ELISA is not suitable for detecting type II collagen fragments in serum. And present two problems. First, perhaps only low levels in the systemic circulation Lacks the sensitivity required for detection of fragments present at Second, ELISA pre Can bind to heat denatured collagen used to coat the wells of the plate Be hindered by some protein.   Therefore, an alternative for collagen fragments that avoids the above problems Need for immunoassay.Description of the invention   Type II collagen molecules are composed of regions with dramatically different susceptibility to hydrolysis Has now been discovered. In particular, amino acids in the triple helix of type II collagen α1-chain Amino acids that fall within the terminal bridging region (residues 201-308) are identified by the remainder of the α1-chain as MM It has now been found that it is very susceptible to hydrolysis by P. Conversely, three layers The carboxyl-terminal cross-linking region (residues 1130-1214) is not very stable, It is reflected in a lower melting point than the amino terminus of this molecule (so it is more subject to hydrolysis Easy to use).   Furthermore, surprisingly, collagen released from degraded type II cartilage in vivo Gen fragment (hereinafter C-II_freeTwo different epitopes Comprising a fragment large enough to have Fragments have half-lives in various body fluids (including serum, urine and synovial fluid) High enough concentration to allow detection and / or accurate measurement by assay It has now been found that it is long enough to allow for an increase.   Furthermore, these epitopes allow the cognate antibody to independently and simultaneously It has also been found that they can be combined. Therefore,the sameC-II_freeOn the fragmentDifferent Become It is now possible to generate two different antibodies to the epitope, Therefore, for the first time, free (eg, serum) type II collagen fragment Dedicated ELISA becomes possible.   Sandwich assays are inherently more sensitive than inhibition assays, and No interference from collagen binding proteins.   Thus, according to the present invention, a first antibody that binds to a first epitope (eg, For example, monoclonal antibodies, polyclonal antibodies, or fragments thereof Is a derivative) and a second antibody (eg, a monoclonal antibody) that binds to a second epitope. Null antibodies, polyclonal antibodies, or fragments or derivatives thereof). Kits (eg, immunoassay kits) are provided, wherein the first and second And the second epitope are collagen membranes released from degraded cartilage in vivo. Fragments (eg, type I or type II collagen fragments) This is the epitope that was pulled. Preferably, the first and second epitopes are C-II_freeThe coupled epitope present above.   As used herein, the term C-II_freeFor example, various diseases or disorders In vivo release as a result of cartilage degradation associated with harm (eg, OA and RA) Any type II collagen fragment released from degraded cartilage by Is included.   Therefore, C-II_freeIs the collagen fragment that enters the systemic circulation (hereinafter -I I_systemic). Such fragments can be found in serum, urine, and synovial fluid. (The latter is hereinafter abbreviated as SF).   As used herein, the term “coupled epitope” refers to a single Antigenic fragments present in the type II collagen fragment or the analyte Refers to pitope. Thus, the coupled epitope is a single type II Collagen fragment complex or complex type II Two or more associated collagen fragments that together contain One or more conformational epitopes anywhere on a single analyte portion May be included.   In a preferred embodiment, each coupled epitope is one continuous epitope. So that all coupled epitopes have a single type II Present on the lagen peptide fragment. Amino acids of α1 type II collagen chain Coupling comprising or located within a sequence or fragment of the N-terminal region Epitopes, such as the amino acid sequence of its triple helix or N-terminal cross-links Epitope containing or located within a fragment of the selected region Are particularly preferred (see FIG. 1).   In a particularly preferred embodiment, the first epitope is residue 243 of the sequence shown in FIG. The second epitope is located within residues 216-236 of the sequence shown in FIG. Located within.   The coupled epitope can bind cognate antibodies simultaneously, and Antibody bound at one epitope sterically hinders antibody bound at a second epitope (Or may be advantageously arranged so as not to obstruct). Therefore, the cup of the present invention The ringed epitopes can be physically "linked" so that their cognate Isolation or splitting of one of the epitope attendants in binding to the antibody also Effectively sequester or split other (linked) epitopes (while this is Remain available for binding to the cognate antibody).   The coupled epitope is preferably at least 2 amino acids, e.g. For example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, Or separated by 20 amino acids. Preferably, they are steric obstacles to simultaneous antibody binding. In order to reduce harm, at least 3 amino acids (eg, about 6 amino acids) Separated.   Generally, the coupled epitope is independent (non-covalent) to the cognate antibody. Blocking) separated enough to allow binding, but Substantially eliminates the possibility of non-pulling or segregating proteolytic cleavage events Close enough to be. Thus, the coupled epitope is Preferably, for example, they are relatively stable or relatively resistant to proteolysis. Part of a type II collagen molecule that undergoes substantial proteolysis in vivo Not separated into short amino acid sequences. N-terminal part of α1 type II collagen peptide (Eg, the N-terminally crosslinked region of the triple helix) is particularly preferred.   The immunoassay kit of the present invention can be used for therapeutic, diagnostic, prognostic, or candidate drug screening. Can be used for training.   Preferably, the immunoassay kit of the present invention comprises a sandwich immunoassay. Key for use in (eg, for use in a sandwich ELISA) It is. In such an embodiment, the first antibody may be immobilized on a solid support. , And / or the second antibody can be labeled.   In such embodiments, an immobilized primary antibody (or capture antibody) is preferred. Or a monoclonal antibody (eg, a mouse monoclonal antibody) The body is preferably polyclonal (eg, a rabbit polyclonal antibody). And from a different species for the capture antibody. This is a species-specifically labeled anti- The use of Ig antibodies allows to detect bound (unlabeled) secondary antibodies Thus, it eliminates the need for labeled collagen-specific antibodies. This In such embodiments, the anti-Ig antibody is preferably alkaline phosphatase or pea. Labeled with oxidase.   Alternatively, a secondary antibody can be labeled. Preferred labels include radiolabels and bio Chin. If biotin is used, the secondary antibody should be Can be detected by contact with avidin.   When the kit includes an enzyme-labeled reactant, preferably, the kit includes an enzyme-labeled reactant. It further includes a substrate for reacting.   Kit includes standard heat denatured type II collagen or C-II_freeCoupling Also includes standard synthetic peptides with pitopes (eg, AH8 and AH12). I can see. Such a synthetic peptide is a protease between the coupled epitopes. And may include a protease-resistant spacer region. Such synthetic peptides are, for example, solid By phase peptide synthesis or by expression of synthetic genes in recombinant host cells Can be synthesized.   As used herein, the terms AH8, AH9, and AH12 refer to specific peptide Used to refer to the pitope or peptide itself (the meaning is indicated And as described in more detail below). Terms AH8L1, AH9L2, and AH12 L3 refers to rabbit antiserum to AH8, AH9, and AH12 peptides, respectively . The term AH8MAb refers to a monoclonal antibody to peptide AH8. Term Anti-AH8 , Anti-AH9 and anti-AH12 refer to peptides AH8, AH9 and AH12, respectively. Antibody that is raised (or (specifically) reacts with it).   In a particularly preferred embodiment, the first antibody is AH8MAb or AH8L1, and The second antibody is AH12L3. Alternatively, the first antibody can be AH12L3 and And the second antibody can be AH8MAb or AH8L1.   The first antibody also competes for AH8MAb or AH8L1 binding to the cognate epitope. The second antibody may be AH12 to a cognate epitope. It may be an antibody that competitively inhibits L3 binding. Alternatively, the first antibody is cognate The second antibody may be an antibody that competitively inhibits binding of AH12L3 to the epitope, and Is an antibody that competitively inhibits the binding of AH8MAb or AH8L1 to cognate epitopes. Can get.   In another embodiment, the first antibody is the same (or present) as the antibody AH8MAb or AH8L1. Qualitatively the same), but the second antibody may be an antibody Can be an antibody with the same (or essentially the same) epitope specificity as AH12L3 . Alternatively, the first antibody has the same (or essentially the same) epitope as the antibody AH12L3. The second antibody may be an antibody AH8MAb or AH8L1. The antibodies may have the same (or essentially the same) epitope specificity.   Binding agents other than antibodies having functionally similar properties to the antibodies of the present invention may also be those of the present invention. Can be used as described. Therefore, the first and / or second antibodies are required Antibodies with specificity (eg, antibodies AH8MAb, AH8L1, or AH12L3) Binding factors derived from, including, or consisting essentially of the variable region Can be a child. Thus, binding agents for use in the present invention include Fab, Fab ' , Or F (ab ')_TwoVarious antibody fragments, such as fragments, or desired Synthetic proteins and peptides in which a variable region having specificity is modeled on the sequence Is mentioned.   In another aspect, the invention provides a C-II_freeBinds to a coupled epitope Antibodies (eg, monoclonal antibodies, polyclonal antibodies, or their fragments) Or derivatives or derivatives) per se. The antibodies of the present invention may be monoclonal or Can be polyclonal, and is preferably labeled. Preferred labels include Biotin and radioactive labels. The antibodies of the present invention also include (eg, Table of wells or probe or dipstick on crotiter plate Surface, such as a surface).   In addition, the first binding portion that binds to the first epitope and the second epitope Bifunctional heteroantibodies comprising a binding second binding moiety are contemplated by the present invention, wherein Wherein the first and second epitopes are C-II_freeThe coupled epitope You. Such heteroantibodies can be coupled C-II_freeIn the presence of the epitope It forms a high molecular weight complex that is easily detectable, so it can be used in various immunoprecipitation assays. Find application.   A first antibody that binds to the first epitope; and a second antibody that binds to the second epitope. Contacting the sample with a second antibody that binds to the biological sample (eg, C-II in vitro or ex vivo)_freeAssay for (Eg, a sandwich ELISA method) are contemplated by the present invention, wherein The first and second epitopes are C-II_freeThe coupled epitope.   The method of the present invention provides C-II_freeAssaying a biological sample for Of various in vitro or ex vivo methods of treatment, diagnosis, or prognosis Find out.   The present invention also provides a method for measuring the therapeutic activity of a drug, comprising the steps of: Intended for: (a) providing a sample from a subject treated with the drug; And (b) C-II using a kit, antibody / binding agent, or method of the invention._freeNitsu Assaying the sample, wherein C-II of the sample assayed in step (b)_{fre e} Is used as an index of therapeutic activity.   In another aspect, the present invention provides for the treatment using the therapeutic activity measurement process of the present invention described above. Screening a library of candidate therapeutic agents for therapeutic activity. A process for producing a therapeutic agent is provided.   In yet another aspect, the present invention provides a method for producing a therapeutic agent comprising the steps of: (A) by a process for producing a therapeutic agent of the invention. Selecting a chemical formula based on one or more structural features of the therapeutic agent produced by the method And then (b) a therapeutic agent having the formula selected in step (a) (eg, Production or isolation). Can be obtained by this process (also Are obtained). These preferably include anti-arthritic drugs and And MMP inhibitors.   In yet another aspect, the present invention provides a cell expressing the antibody or binding factor of the invention. Or a cell line is intended. The cell or cell line may be prokaryotic or eukaryotic (eg, Mammals). High expressing monoclonal antibodies according to the invention Bridomas are preferred.   In a still further aspect of the present invention, C-II_freeResults in a coupled epitope A process for producing a matching antibody is provided, the process comprising the steps of: Do: (a) Type II collagen in vitro to produce multiple fragments Degrading; (b) fractionating fragments based on size; (c) relatively large Identifying the critical fragment; (d) this ratio for the presence of multiple epitopes. Screening relatively large fragments; (e) two or more epitopes And (f) selecting the fragment selected in step (e). Raising antibodies to the antibodies.   C-II obtainable (or obtainable) by the above process_freeThe Coplin Antibodies that bind to a tagged epitope are also encompassed by the present invention.   The invention also relates to the process of step (f) of the invention, as well as to the isolated fragment of the invention. Antibody or C-II comprising raising an antibody against the fragment_freeBinding factor Has more than one epitope for use in the process for producing And isolated type II collagen fragments.Application   The present invention provides a variety of approaches, including diagnostic, prognostic, and drug screening assays. Find applications in ISSEY. The present invention also provides a therapeutic regimen as described below. Can be used as part of Thus, the present invention provides therapeutic, prognostic, and diagnostic It can be useful in various forms.   For example, in diagnosis, the sandwich ELISA of the present invention can be used to treat urine, serum, Or sufficient sensitivity to detect type II collagen fragments in SF. Trial Can be easily obtained without risk to the patient, and the assay is simple and inexpensive. You. Therefore, the population's daily routine for arthritis or other diseases that result in cartilage destruction Effective screening is possible using the methods and reagents of the present invention.   The present invention also relates to on-going cartilage in patients with different types of OA or RA. It can be used to determine the degree of collagen degradation. Therefore, the assay It also predicts, for example, whether a patient is progressing to develop severe cartilage lesions. To find utility as a basis for prognostic testing.   In therapy, the present invention relates to the in vivo efficacy of anti-arthritic drugs, such as MMP inhibitors. Is provided. Many such drugs have been tested in clinical trials (3,26). Before the present invention, whether patients are protected by treatment Type II collagen degradation using urine, serum, or SF markers to determine There was no way to follow. Thus, the present invention relates to adjuvants for various forms of treatment. To determine the optimal dose and / or frequency of application or therapeutic agent administration May find application as a means of doing so.   Further uses of the assays of the invention include the diagnosis of growth disorders and growth hormones. Testing the efficacy of the treatment or monitoring progress. For example, long bone Growth depends on the progression of endochondral bone, where growth plate cartilage is degraded and replaced by bone (1). Therefore, serum levels of type II collagen fragments are higher than in adults. Even higher in normally growing children reflects this process. But growth impairment In children of all ages, the serum levels of these fragments are higher in children of any given age. Seems to be lower than normal. Therefore, the present invention provides a dose-response To establish relationships and to monitor the progress of the disorder and its treatment It can be used to diagnose such disorders as an adjunct to drug treatment.   The present invention has now been described with respect to several embodiments, which are for illustrative purposes only. Yes, and is not limited in any way.   The examples refer to some drawings:   FIG.Identifies different molecular bands or domains in the type II collagen α chain, and antibodies. Figure 3 schematically illustrates the location of the epitope used to generate.     1: Epitope AH12     2: Epitope AH8     3: Epitope CB11B     4: Triple helical area     5: Collagenase cleavage site     6: Helical bridge     7: Epitope AH9     8: carboxy terminal propeptide     9: carboxy-terminal telopeptide     10: Telopeptide cross-linking     11: Carboxy-terminal cross-linked region     12: Helical bridge     13: triple helix amino-terminal cross-linked region     14: Amino-terminal telopeptide     15: amino terminal propeptide     16: Telopeptide crosslinking   FIG.Shows the primary structure (amino acid sequence) of human type II collagen α chain.   FIG.Outlines inhibition and sandwich ELISA assay formats Is shown.     Panel A:Inhibition ELISA       1: The type II collagen fragment in the solution phase binds to the primary antibody and Inhibits it from binding to collagen.       2: Primary anti-collagen antibody binds solid phase denatured collagen.       3: heat-denatured type II collagen coated on a solid phase.       4: Enzyme-labeled second-step antibody binds to primary antibody.Panel B:Sandwich ELISA       1: Enzyme-labeled avidin binds to biotin.       2: The type II collagen fragment in the solution phase is a collagen fragment Binds to the primary antibody via the first epitope above.       3: Primary anti-type II collagen antibody coated on solid phase.       4: Biotinylated secondary anti-type II collagen antibody is a collagen fragment Binds to a second epitope on the   FIG.Indicates characterization of antiserum AH8L1. Antiserum AH8L1 was ligated to peptide AH8 (panel (A) and HDC (Panel B). The result Specific antiserum (○) compared to preimmune serum (◇) for one antigen You.   FIG.Indicates characterization of antiserum AH9L2. Antiserum AH9L2 was converted to peptide AH9 (panel (A) and HDC (Panel B). The result Shows specific antiserum (○) compared to preimmune serum (◇) for one antigen You.   FIG.Indicates characterization of antiserum AH12L3. Antiserum AH12L3 was conjugated to peptide AH12 (P Tested in ELISA assays against panels A) and HDC (panel B). The result Specific antiserum (○) compared to preimmune serum (◇) for both antigens Show.   FIG.Shows cross-reactivity of anti-peptide antisera with different collagens. A) Ku B) rabbit anti-peptide AH8L1 antiserum; C) rabbit anti-pep Pide AH9L2 antiserum; D) Rabbit antipeptide AH12L3 antiserum. All three panels Lane 1 is human type 1 collagen and lane 2 is bovine type I collagen Yes, lane 3 is human type II collagen, lane 4 is bovine type II collagen And lane 5 is human type III collagen and lane 6 is bovine type III collagen And lane 7 is human type IV collagen.   FIG.Shows a Western immunoblot of bovine chondrocyte lysate. Rice Plates were added with bovine type II collagen (lane 1) or without (lane 2). ). Panel A shows the lysate after transfer to nitrocellulose. 7 is amide black staining of total proteins in the sample. Panels B, C, and D Western immunoblot using antibodies AH8L1, AH9L2, and AH12L3, respectively It is. The movement position of the type II collagen α chain is shown.   FIG.Shows a Western immunoblot of 3-week media from bovine nasal cartilage culture. . Medium from IL-1 stimulated cartilage cultures was separated by SDS-PAGE on a 15% gel. , And it electroblotted on the nitrocellulose membrane. Panel A is a nitrocell Amide black staining of total protein in lysate after transfer to loin Indicates color. Panels B, C, D, and E show antibodies Pa-3 / 4, AH8L1, AH9L2, respectively. And Western immunoblot with AH12L3. The two most clearly defined The migration position of the extracted collagen fragment, as well as the molecular weight standard are shown.   FIG.Is the position of the amino terminus of fragment TII-F1 in the type II collagen triple helix. Position.   FIG.Indicates a double bond of AH8L1 and AH12L3 to HDC. Purified biotinylated anti The body AH8L1 was treated with antiserum AH12 on itself (（in both panels) and at a 1: 100 dilution. With L3 (in panel A) or with antibody AH12L3 purified at a 1:64 dilution (in panel B) Ii) It bound to HDC after HDC pre-incubation. AH8L1 binding Avidin-peroxidase binding method.   FIG.Shows a comparison of the type II collagen assay. HDC with the sandwich of the present invention. H (panel A) or the known CB11B inhibition ELISA (panel B). Assayed over a range of standard concentrations.   FIG.Shows immunohistochemical staining of human OA femoral head cartilage from a human subject. Dye Colors were determined using mouse monoclonal antibody Col2-3 / 4m (A and D), rabbit polyclo Null antibody AH12L3 (B and E) and rabbit polyclonal antibody AH9L2 (C and And F). Negative controls (non-immune IgG) were added to mice (G) and And rabbit (H) are shown separately.   Fig. 14Shows immunohistochemical staining of human normal femoral head cartilage from a human subject. Staining was performed using mouse monoclonal antibody Col2-3 / 4m (A) and rabbit polyclonal antibody. AH12L3 (B) and rabbit polyclonal antibody AH9L2 (C). negative A positive control (non-immune IgG) was used for mice (D) and rabbits (E). Shown separately.   Fig. 15Shows double binding of antibodies AH8MAb and AH12 to HDC. Purified antibody AH12 On itself (■) or after preincubation of HDC with AH8MAb (●) Goat anti-mouse bound to HDC and labeled with alkaline phosphatase Detected using a secondary IgI antibody. Goat anti-mouse labeled with alkaline phosphatase Binding of monoclonal AH8MAb on itself, detected by secondary Ig antibodies , Are shown for comparison (（).   FIG.Shows AH8MAb / AH12L3 sandwich ELISA standard curve. The results are Averaged for 11 separate experiments using type II collagen purified from septal cartilage Shown as +/- SEM.   Fig. 17Characteristics of denatured type II collagen by AH8MAb / AH12L3 sandwich ELISA 2 shows heterogeneous detection. Native bovine type II collagen was prepared as described in Example 5. And then partially denatured. Samples were then assayed in a series of dilutions.   Fig. 18Indicates a type II collagen fragment in normal human serum. Serum for men (N = 3) and female (n = 5) volunteers (age range 23-33) And assayed at various dilutions using AH8MAb / AH12L3 sandwich ELISA. Each panel shows the results for one individual.   FIG.Shows estimates of type II collagen fragment levels in normal human serum. Serum from male (n = 3) and female (n = 5) volunteers (age range 23-33) And assayed using an AH8MAb / AH12L3 sandwich ELISA. Ko Lagen fragment concentration values were determined using a heat denatured bovine type II collagen standard curve. Was estimated.   Fig. 20Is a standard synthetic peptide for use in the AH8MAb / AH12L3 sandwich ELISA. IndicatesExample 1-Identification of immunogenic epitopes, peptide synthesis, and carrier proteins Coupling to quality   Amino acid sequences from the amino and carboxy terminal bridging regions of the α1 (II) chain Aqueous analysis and appropriate immunogenic peptides were identified using the criteria of (10) above. Selected.   Amino-terminal cysteine for coupling to carrier proteins Peptides AH8, AH9 and carboxy-terminal tyrosine for efficient iodination And AH12 were synthesized respectively. As shown in FIG. 1, AH8 and AH12 AH9 is located in the amino-terminal cross-linking region, and AH9 is You. Peptides were purchased from Dr. Kreb's Institute, University of Sheffield. Arthur Mo synthesized by ir, and the sequence is as follows:   Each of these peptides is coupled with the coupling reagent bromoacetic acid-N-hydroxy Using the succinimide ester (Sigma), its amino-terminal cysteine residue Via the carrier protein keyhole limpet hemocyanin (Calbioch em, Nottingham, UK). The method of conjugation is based on peptide binding to ovalbumin. The coupling was exactly as described (10).Example 2-Antibody production   Adult New Zealand white rabbits were each 500 μg peptide conjugated to KLH. And immunized by subcutaneous injection. Two animals were immunized using each peptide . For the first immunization, the antigen is emulsified with Complete Freund's adjuvant (Sigma). Was. Subsequent immunizations are two weeks apart and incomplete Freund's adjuvant (Sigma) was used. Pre-immune and specificity test sera are used as described below. Next, the cells were screened for reactivity with type II collagen.Example 3-Antibody Screening ELISA   Heat-denatured type II collagen (HDC) is converted to bovine type II collagen (Sigma) using 0.1M charcoal. Acid buffer (0.1M NaHCO_Three, 0.1M Na_TwoCO_Three, PH 9.2) and 20 minutes at 80 ° C Prepared by heating. Immulon-2 ELISA plate (Dynatech Labs, Guernsey, UK) with 2 μg HDC per well by passive adsorption at 4 ° C. for 72 hours. Coated. Plates were washed with PBS containing 0.1% v / v Tween-20 (Sigma; PBS- Tween), then 1% w / v bovine serum albumin in PBS (BSA, Sigma) And blocked by incubation at room temperature for 30 minutes. Plate, PB Wash once with S-Tween, and add rabbit antiserum to ELISA plate Was added.   After incubation at 37 ° C for 90 minutes, the plate is washed three times with PBS-Tween And then alkaline phosphatase labeling diluted 1: 1000 with PBS-Tween-1% BSA Goat anti-rabbit immunoglobulin (Southern Biotech, Alabama, USA) Was added to the well. Plates are incubated for 90 minutes at 37 ° C., then PBS-Tw Washed three times with een and once with distilled water. Alkaline phosphatase substrate p-d Disodium trophenyl phosphate, 9.0 mM diethanolamine, 0.25 M MgCl_Two, Prepare fresh at a concentration of 0.5 mg / ml in pH 9.8 and add each well for 20-30 minutes at room temperature. Was added to the well. Absorbance measured at 405 nm on a Dynatech Multiscan plate reader It was measured.Example 4-SDS-PAGE and Western immunoblotting   SDS-PAGE was performed as described previously (10) under reducing conditions at 7.5%, 10%, and Was performed on 15% miniprotean gel (Bio-Rad Labs, Hertfordshire, UK). Gel 0.2% w / v Coomassie Brilliant Blue in 50% v / v methanol, 10% v / v acetic acid ( Bio-Rad) and destained with 20% v / v methanol, 5% v / v acetic acid. Go In some cases, electrophoresed samples were washed with Tris containing 10% or 20% v / v methanol. transfer to nitrocellulose membrane in s-glycine buffer, and Prior to immunoblotting with antibodies Pa-3 / 4, AH8L1, AH9L2, or AH12L3, PB Block overnight with S-3% BSA at room temperature.   Each of the blocked membranes was diluted with appropriate dilutions in PBS-3% BSA. And incubated for 2 hours at room temperature. After three washes with PBS-Tween, The membrane was conjugated with alkaline phosphatase, diluted 1: 1000 with PBS-3% BSA. Goat anti-mouse (for Pa-3 / 4) or anti-rabbit (for AH8, 9, and 12) ) Incubated with secondary antibody for 30 minutes at room temperature. Membrane with PBS-Tween Washed three times and once with distilled water. Alkaline phosphatase substrate solution Lomo-4-chloro-3-indoyl phosphate and nitro blue tetrazolium Prepared from the kit (Bio-Rad). Add this to the membrane and Incubated at room temperature until the appropriate color developed. Rinse the reaction with distilled water Stopped by that.Example 5-Bovine nasal cartilage culture   Freshly excised cartilage from the bovine nasal septum is cut into 5 cm x 1.5 cm x 2 mm slices with a scalpel I cut it. Slices were washed once with sterile PBS for 20 minutes. Cartilage disc (40mg Average wet weight) was prepared using a sterile stainless steel punch. Cartilage sura The outer part of the chair was not used to avoid contamination with other tissues.   Nasal and articular cartilage explants were treated with glutamine (2 mM), penicillin G (2000 U / ml) And streptomycin (0.1 mg / ml) and 4- (2-hydroxyethyl) -1-pi Serum-free solution containing perazineethanesulfonic acid (HEPES, 10 mM) (all from Gibco) Rubecco's modified Eagle's medium (DMEM; Gibco Life Technologies Ltd., Renfrew Ro ad, Paisley, UK) in a 48-well tissue culture plate (Costar, High Wycombe, U.S.A.). K) for 4 weeks, 5% CO_TwoThe cells were cultured at 37 ° C. in a humid atmosphere of / 95% air. Each way The kit contained two disks in 400 μl of medium. Insert the disk with Dr. Michael B . Recombinant human IL-1α, courtesy of Widmer, Immunex Corp., Seattle, USA Processed. On days 7, 14, and 21, the media in each well containing fresh IL-1α was Refilled.Example 6-amino terminal sequencing   Media samples and free radical scavengers for amino-terminal sequencing Run on a pre-run gel and then adjust the pH as described in Dunbar and Wilson (27). Buffer exchange was performed for correctness. Transfer the sample to a PVDF membrane (Bio-Rad). Troblot and 0.5% w / v Coomassie in 7% v / v acetic acid, 35% v / v methanol Stain with Brilliant Blue R-250 for 10 minutes, and remove background The mixture was decolorized with 7% v / v acetic acid 25% methanol until complete. Cut off relevant protein bands And Bryan Dunbar, Department of Molecular and Cell Biology, Un Analyzed by iversity of Aberdeen using automated sequencing equipment .Example 7-Affinity purification and biotinylation of antibodies   The antibody is purified and the antigen is purified according to the newly described method of Rucklidge et al. (28). Biotinylation was performed using a blotting membrane as a support. AH8L1 and And AH12L3 antibodies, both with HDC coated PVDF membrane (Bio-Rad) Was used for affinity purification from immunized rabbit serum. AH8L1 antibody Prior to stripping from the run, biotin (biotinamide) in 0.1 M carbonate buffer Caproate N-hydroxysuccinimide ester, Sigma) at 75 μg / ml for 30 minutes Between biofilms while still bound to the membrane. It has become. Both purified antibodies are tested for yield and specificity before use. I did it.Example 8-Sandwich ELISA assay for type II collagen fragment   A 1: 8 dilution of the capture antibody (affinity-purified AH12L3) in 0.1 M carbonate buffer Was passively bound to Immulon 2 plates for 1 hour at 37 ° C. Then, all web Were blocked with carbonate buffer containing 0.2% BSA for 1 hour at 37 ° C, then PBS / Twee Washed three times with n. HDC is diluted in PBS, added to appropriate wells, and Incubated for 0 minutes.   After three additional washes with PBS / Tween, the antibody to be detected (biotinylated AH8L1) was Add to all wells at a 1: 8 dilution in S / Tween / 0.1% BSA and for an additional 90 minutes Incubated. Plates are washed three times with PBS / 0.1% Tween, then PBS / Twee Incubate with avidin-peroxidase (Sigma) at 20 mg / ml for 30 min at room temperature. And then washed three more times with PBS / Tween and once with distilled water. 0.1M Na_TwoH PO_FourAnd 10 μl H_TwoO_TwoAt a concentration of 0.5 mg / ml in 0.05 M citric acid containing Zease substrate (o-phenolenediamine dihydrochloride, Sigma) was added to each well and 0M H_TwoSO_FourThe color was allowed to develop for 20-30 minutes prior to the stop. The absorbance was read at 490nm.Example 9 Characterization of Rabbit Antiserum Against Peptides AH8, 9, and 12   Each peptide was used to generate antisera in two rabbits. All three pairs For peptides, both antisera are similar to each other with respect to titer and specificity. Worked essentially.   In the ELISA assay, antibody AH8L1 reacted specifically with peptides AH8 and HDC (FIG. 4) did not react with peptide AH9 or AH12 (data not shown). Anti The body AH9L2 reacted specifically with the peptides AH9 and HDC (FIG. 5), but the peptide AH8 Or did not react with AH12 (data not shown). Antibody AH12L3 is a peptide AH12 And HDC (Figure 6), but did not react with peptide AH8 or AH9 (Data not shown). AH9L2 detects type II collagen in an ELISA assay Were clearly less potent than AH8L1 or AH12L3 (FIGS. 4B, 5B, and 6 Compare B).   Antibodies AH8L1, AH9L2, and AH12L3 were combined with various types of heat-denatured collagen. Was tested by Western immunoblotting. It Each of them recognizes only type II collagen and type I, III, or IV collagen. AH9L2 did not recognize this gene (Fig. 7), but AH9L2 Method was not effective in detecting type II collagen.   As further proof of specificity, each antibody can be used to determine the number of proteins present in The proteins were tested by Western immunoblot. These proteins Were not detected by any of the antibodies, but these were added to the lysate. Type II collagen was detected (FIG. 8).Example 10-Type II collagen fragment in culture medium from bovine nasal cartilage culture Detection   When bovine nasal cartilage is stimulated with 50 ng / ml IL-1α, there is a large increase in MMP activity in the medium. Type II collagen fragmentation into the medium during the third week of culture, coincident with the addition of There is a sudden release of the object (14). The medium of the third week is used for such a highly degraded culture. From the sample, and any of the type II collagen fragments were stained with 10% SDS-PAGE gels. Western im knob to determine if it is large enough to appear on the le Inspected by lotting.   Using an inhibition ELISA for CB11B, large amounts of this epitope were present in the media samples. (Not shown). Many protein bands are present in the week 3 culture medium. (FIG. 9A). But perhaps all of the fragments containing the epitope CB11B None of these with the antibody Pa-3 / 4, as was small and migrated from the bottom of the gel It was not detected by Western immunoblot (FIG. 9B).   Conversely, a number of fragments were analyzed by Western immunoblotting with antibody AH8L1. And two of the fragments, TII-F1 and TII-F2, are each approximately 28 kD Appeared as sharp, well-defined bands with molecular weights of A and 27 kDA (FIG. 9C) . Antibody AH9L2 did not detect type II collagen fragment (Fig. 9D), Body AH12L3 detected the same band as AH8L1 (compare FIG. 9E with 9C).   Similar results were obtained in each case using four other media using media from different cartilage cultures. Obtained experimentally (not shown). Therefore, type II collagen by Western blot Only the antibody to detect the fragment is the amino-terminal helix of the α1 (II) chain. Antibody to an epitope in the protein region.   Antibodies AH8L1 and AH12L3 bind type II collagen fragments in these cultures. It was important to have detected and proved not to cross-react with contaminants . For this purpose, the most abundant of the two sharp bands detected by the antibody Of TII-F1 from the PVDF membrane and the first of its amino-terminal sequence Of 16 residues were determined. The sequence has an initial yield of 17.2 pmol and the α1 (II) chain 100% matched for internal sequence. This is because TII-F1 is actually At residue 223 within the sequence of peptide AH12. This proved that it was located at the amino terminus (FIG. 10). Therefore, antibody AH8L 1 and the epitope detected by AH12L3 are the major detectable fragments Located very close to the amino terminus ofExample 11-Type II collagen fragment using antibodies AH8L1 and AH12L3 Of sandwich ELISA   Epitopes AH8 and AH12 are separated by exactly 6 residues in the α1 (II) chain So both antibodies rewind simultaneously without causing steric hindrance to each other It was important to determine if it would bind to the α-chain.   For this purpose, the ELISA plate is coated with HDC, blocked and then Conjugated unlabeled antibody AH12L3 to collagen as either antiserum or purified antibody. I combined. The biotinylated antibody AH8L1 is then added to the wells and the binding is Detection was performed using an avidin-peroxidase conjugate. The result (Fig. 11) is AH12L 3 did not inhibit the binding of biotinylated AH8L1 to HDC.   Preliminary sandwich ELISA, unlabeled antibody AH12L3 coated on ELISA plate , Incubating with HDC, then biotinylated antibody AH8L1 By detection with avidin-peroxidase conjugate. HDC , Detected by this assay, and the lower limit of detection is about 0.05 μg / ml (Figure 12A).   By comparison, the CB11B inhibition ELISA yields type II collagen with a detection limit of 50-100 μg / ml. Was detected (FIG. 12B).Example 12: Immunostaining of osteoarthritis cartilage (i)Immunohistochemistry   Cartilage pieces were placed in OCT embedding medium (B.D.H. Laboratory Supplies, Poole, England). And excise 7 μm thick sections and 3-aminopropyl as described (11). Purify with ropirtriethoxysilane (I.C.N. Biomedicals Ltd., Thame, England) Attached to recoated slides to ensure maximum attachment of sections for uniform staining Sure. Sections are either stained immediately or stored at −20 ° C. there were. Aldehyde groups are blocked with normal goat serum diluted 1:10 and cells Chondroitiners containing proteinase inhibitors Maximized with ABC ABC lyase (I.C.N.). Sections should be Col2-3 / 4m, AH9L2, or AH1 Probed with 2L3. Adjust the optimal dilution to the appropriate IgG concentration for the control antibody. Determined by matching with degree. The same for IgG1 Col2-3 / 4m Mouse monoclonal antibody raised to a sotype but unrelated epitope It was used. About IgG purified from rabbit anti-peptide antisera AH9L2 and AH12L3 Used control IgG (Vector Laboratories) from non-immune rabbits . These control antibodies produce negligible background staining of cartilage specimens. Diluted to the highest concentration. A biotin-avidin detection system was used. Section 1 : 100 diluted biotin-labeled secondary antibody, goat anti-rabbit or mouse Ig (H + L) (Southern Biotechnology Associates). Join Antibody to ABC Elite Biotin-Streptavidin Kit according to manufacturer's instructions (Vector Laboratories) (avidin-biotin-peroxidase) The peroxidase reaction is then nickel to form a gray-black stain. Use 3,3'-diaminobenzidine (DAB) (Vector Laboratories) And it developed color. Sections are graded before permanent implantation with DPX (B.D.H.) Dehydrated with alcohol. (ii)result   Samples from a variety of different osteoarthritis and non-arthritis joints were analyzed. All trials The material was stained with COL2-3 / 4m, AH12L3, and AH9L2. Fig. 13 shows the OA femoral head cartilage Here is a tabular example. There were differences in the intensity and pattern of staining between the three antibodies. Ma The results with us monoclonal Col2-3 / 4m were essentially as described (11). Staining is seen throughout the depth of the OA cartilage, with thick bands of strong staining on the joint surface And there was moderate staining extending to the central zone (FIG. 13A). Deep zone chondrocytes are strong Pericellular staining was shown (FIG. 13D). Some extent of interregional staining was Was seen in Non-immunized mouse controls are shown in FIG. 13G for comparison. Rabbit Polyclonal antibody AH12L3 gives very similar results to Col2-3 / 4m but stains The intensity was weaker (FIGS. 13B and E). In contrast, rabbit polyclonal antibody AH Staining at 9L2 was generally very weak in all bands (FIGS. 13C and F). this The intensity of the staining with the antibody is only slightly greater than the staining with the non-immune control. It was hard (Fig. 13H).   Generally, staining of non-arthritis cartilage was much less intense. Figure 14 shows non-arthritis A representative example of femoral head cartilage is shown. The pattern of staining was similar to that described previously (11) . Interregional staining seen with Col2-3 / 4m and AH12L3 was of low intensity and As limited to bands stained on the joint surface (FIGS. 14A and B). Chondrocytes I The degree and intensity of staining was also greatly reduced. Same as non-immune control In most cases at the bell, in fact, there was a complete absence of staining with AH9L2 ( (Figure 14C).   Therefore, the amino-terminal epitope AH12 was identified by immunohistochemistry on OA cartilage. It can be seen that the detection did not detect the carboxy terminal epitope AH9 You. This means that the amino-terminal region degrades more gradually and thus in tissue Suggests a longer retention. This is the phase compared to the carboxy terminal region. Further evidence for stability.Example 13: Production and characterization of monoclonal antibodies against the AH8L1 epitope   Monoclonal antibodies were purified from AH8E for use with AH12L3 in a sandwich ELISA. Raised against the pitope. BALB / c mice received 1 injection with KLH-conjugated AH8L1 Immunized subcutaneously five times with 100 μg of peptide. First injection with complete Freund's horse mackerel Emulsified with Couvant, subsequent injections were in incomplete Freund's adjuvant. Spleen cells from all of the mice were pooled, fused to SP2 / 0 myeloma cells, and Monoclonal antibodies were prepared using conventional techniques. Positive claw Based on the ability of the secreted antibody to react with AH8 and HDC by ELISA. (As described above, goat anti-mouse Ig-alkaline phos Phatase (using Southern Biotechnology)). Copy the selected clone to Sh effield Hybridoma Unit, Spinner frus at Sheffield University Expanded throughout The resulting crude antibody preparation is applied to a Thiosorb column (Bioprocessing , Durham, UK) to purify the IgG. Isotype of the resulting preparation With the Isostrip Mouse Monoclonal Antibody Isotyping Kit (Boehringer Mannheim ) Was used to determine as IgG1. After dialysis against PBS, the concentration of the preparation was increased to 280 nm. Determined by reading the absorbance of (2.15 mg / ml) and aliquots at -20 ° C Saved in.   Experiments also showed that the binding of AH8MAb to an epitope on HDC To ensure that AH12L3 binding to its corresponding epitope is not inhibited I went to. AH12L3 and AH8MAb, HDC coated Immulon-2 plate row Dilute each with carbonate buffer (see methods) across and at 37 ° C. For 90 minutes. Remove unbound antibody from PBS-Tween And removed by three washes. The double diluted AH12L3 was then combined with the AH8MAb already Add to one of the incubated rows and incubate at 37 ° C for a further 90 minutes. I did it. PBS alone was added to the remaining rows. After washing, secondary antibody is added; AH8MA b only and anti-mouse against AH8MAb + AH12L3 row, and against AH12L3 only row Anti-rabbit, and incubated as above. After final cleaning, alkaline Add phosphatase substrate to all wells and read plate at 450 nm It is.   AH8MAb found to be specific for AH8 peptide and HDC by ELISA However, it shows minimal cross-reactivity with AH12 and AH9 peptides. AH8L1 polyc We also see that lonal antibodies do not bind to type I, III, or IV collagen. Collagenase cleavage as indicated by western blotting 3/4 pieces of the type II collagen alone were positive. AH8MAb also Week 3 media from the same IL-1α stimulated BNC as polyclonal AH8L1 and AH12L3 A middle band was detected (data not shown).Example 14: AH8MAb / AH12L3 sandwich ELISA (i)Preparation   All wells of Immulon-2 plate were 1:50 at 50 μl of AH8 MAb (27 μg / ml) Coated and antibodies were passively adsorbed overnight at 4 ° C. Excess antibody It was removed by washing three times with azide-free PBS containing 0.1% Tween 20 (PBS-Tween). Co Unbound binding sites were replaced with 5% w / v skim milk powder in PBS (Marvel^TM)so Blocked for 30 minutes at room temperature, then washed three times with PBS-Tween. 50 μl PBS Added to 4 wells to determine the degree of non-specific binding. Purified natural or denatured Collagen was added to duplicate wells. Natural collagen into 0.5M acetic acid First dissolved and then dialyzed against 0.5 M Tris HCl. Then, some Aliko The plate was denatured by heating at 80 ° C. for 20 minutes. Dilute with PBS if necessary Serum samples were added in duplicate to test wells at 50 μl / well. Then, Rates were incubated at 37 ° C. for 90 minutes before 3 washes with PBS-Tween . The secondary antibody AH12L3 was diluted 1: 500 in PBS containing 0.1% Tween 20 and 3% BSA, Then 50 μl was added to all wells. Incubate further at 37 ° C for 90 minutes After incubation, the plates are washed as above and the tertiary antibody peroxidase -Conjugated goat anti-rabbit Ig (Sigma Biosciences) diluted 1: 1000 in PBS-Tween-BSA Add 50 μl / well to all wells and incubate at 37 ° C for 90 minutes. I did it. After three additional washes with PBS-Tween and a final wash with distilled water, The oxidase substrate TMB-3,3 ', 5,5'-tetramethylbenzidine (Kirkegaard and Per ry Laboratories, Gaithersburg, Maryland, USA) at 50 μl / The wells were added and the color allowed to develop for 20-30 minutes at room temperature. Reaction 50 μl H_Two SO_FourWas stopped and the plate was read at 450 nm. Four non-specific binding webs The average absorbance of all other wells was subtracted from the absorbance of all other wells. (ii)AH8MAb / AH12L3 Antibody characterization for sandwich ELISA   Epitopes AH8 and AH12 are located close to each other, and H8MAb and polyclonal AH12L3 convert denatured type II collagen simultaneously It was important to show that both could bind. Figure 15 is already joined Demonstrates the ability of AH12L3 to bind to an epitope on HDC in the presence of AH8MAb. This Curve is almost identical to the curve produced when AH12L3 bound to HDC alone. It can be seen that there is no decrease in AH12L3 binding. This is the AH8 and AH12 epi The topes are close to each other, but both antibodies do not interfere with each other, Indicates that it can bind to the tope. This is for assays performed with sandwich ELISA. If the type II collagen flag with these epitopes in serum or synovial fluid Enables the detection of a statement. (iii)AH8MAb / AH12L3 Establishment of a standard curve for sandwich ELISA   Standard curve when both HDC concentration and absorbance are plotted on a log scale Achieved a high degree of linearity (Figure 16). Ensure that the sample is within the limits of the standard curve Dilute appropriately. (iv)Characteristics of AH8MAb / AH12L3 sandwich ELISA for denatured type II collagen Opposite sex   The assay results for native and denatured type II collagen are shown in FIG. As expected, type II collagen was detected only in its denatured form, Not detected in the case of the application. In separate experiments, natural or denatured I and III Type A collagen was assayed using an AH8MAb / AH12L3 sandwich ELISA, There was no recognition of these molecules in the assay (data not shown).Example 15: Measurement of type II collagen in serum   Collect various sera from non-arthritic laboratory personnel and store at -20 ° C until use did. These were added to the new AH8MAb / AH12L3 sandwich ELI over a range of dilutions. Each was assayed for type II collagen using SA. The results shown in FIG. The fragment of type II collagen containing AH8 and AH12 epitopes And the detected levels could be diluted in a log-linear fashion Indicates that Type II collagen fragment in normal serum compared to bovine HDC standard Levels ranged from 0 to about 600 μM, with an average of about 100 μM (FIG. 19).Example 16: Preparation of synthetic peptide standard   Purified bovine type II collagen can be used as a standard for the assay However, there may be batch-to-batch variation in this standard. Thus, epitope AH8 A 50 residue peptide containing both AH12 and AH12 was synthesized. The sequence is shown in FIG. This mark Jun monitors disease-related changes in serum levels of type II collagen fragments Can be used in assays designed to perform

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 21/08 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 33/68 33/68 C12N 5/00 B (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF , BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ , D E, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT , LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Clocher, Lisa Jane United Kingdom Es 10 2 Cue Cue Saus Yorkshire, Sheffield, Bloomhill, Westbourne Road 34

Claims (1)

[Claims] 1. A first antibody (eg, a monoclonal antibody, a polyclonal antibody, or a fragment or derivative thereof) that binds to a first epitope and a second antibody (eg, a monoclonal antibody, a polyclonal antibody, or a fragment thereof) that binds a second epitope Or a derivative thereof, wherein the first and second epitopes are C-II _free coupled epitopes. 2. The immunoassay kit of claim 1 for use in therapy, diagnosis, prognosis, or candidate drug screening. 3. An immunoassay kit according to claim 1 or 2 for use in a sandwich immunoassay (eg for use in a sandwich ELISA). 4. The method according to any one of claims 1 to 3, wherein the C-II _free is systemic C-II _free (for example, serum C-II _free , urine C-II _free , or synovial fluid C-II _free ). Immunoassay kit. 5. 4. The method of claim 1, wherein the coupled epitope comprises (or is located within) an amino acid sequence from the N-terminal region of the α1 type II collagen chain (eg, the N-terminal bridging region of the triple helix). 5. The immunoassay kit according to any one of 4. 6. 6. The method of any of claims 1 to 5, wherein the coupled epitope is capable of binding cognate antibodies simultaneously and / or does not sterically hinder the antibody bound at the first epitope at the second epitope. The immunoassay kit according to claim 1. 7. The immunoassay kit according to any one of claims 1 to 6, wherein the coupled epitope comprises a conformational and / or continuous epitope. 8. The coupled epitope comprises at least 2 amino acids, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 The immunoassay kit according to any one of claims 1 to 7, which is separated by amino acids. 9. The immunoassay kit according to any one of claims 1 to 8, wherein the first antibody is immobilized on a solid support and / or the second antibody is labeled. 10. The immunoassay kit according to claim 9, wherein the label is biotin. 11. The immunoassay kit according to claim 9, wherein the label is a radioactive label or an enzyme label (for example, alkaline phosphatase or peroxidase). 12. The immunoassay kit according to claim 10, further comprising enzyme-labeled avidin. 13. The first antibody is an anti-AH8 antibody (eg, AH8MAb or A8L1) and the second antibody is an anti-AH12 antibody (eg, AH12L3), or the first antibody is an anti-AH12 antibody (eg, AH12L3) and the second antibody is an anti-AH8 antibody (e.g., AH8MAb or AH8L1). 14． The first antibody is an antibody that competitively inhibits binding of AH8MAb or AH8L1 to a cognate epitope, and the second antibody is an antibody that competitively inhibits binding of AH12L3 to a cognate epitope, Alternatively, the first antibody is an antibody that competitively inhibits binding of AH12L3 to a cognate epitope, and the second antibody is an antibody that competitively inhibits binding of AH8MAb or AH8L1 to a cognate epitope. An immunoassay kit according to any one of claims 1 to 12. 15. The first antibody is an antibody having the same (or essentially the same) epitope specificity as the antibody AH8MAb or AH8L1, and the second antibody is the same (or essentially the same) epitope as the antibody AH12 L3 An antibody having specificity, or the first antibody is an antibody having the same (or essentially the same) epitope specificity as antibody AH12L3, and the second antibody is an antibody AH8MAb or AH8L1 The immunoassay kit according to any one of claims 1 to 12, which is an antibody having the same (or essentially the same) epitope specificity as described above. 16. The said 1st and / or 2nd antibody is a binding factor containing the variable region (or functional derivative thereof) of antibody AH8MAb, AH8L1, or AH1 2L3 (for example, Fab or Fab 'fragment), The binding factor. 13. The immunoassay kit according to any one of 12. 17． An antibody that binds to a C-II _free coupled epitope (eg, a monoclonal antibody, a polyclonal antibody, or a fragment or derivative thereof). 18. 18. The antibody of claim 17, wherein the antibody is labeled (e.g., with biotin or a radiolabel) and / or immobilized (e.g., on a solid support). 19. A kit according to any one of claims 1 to 16, a method according to any one of claims 26 to 28, or for use in a process according to any one of claims 29 to 33. The antibody according to claim 17 or 18. 20. Anti-AH12 antibody (eg, AH12L3) or anti-AH8 antibody (eg, AH8MAb or AH8L1). 21. An antibody that competitively inhibits binding of AH8MAb, AH8L1, or AH12L3 to a cognate epitope. 22. An antibody having the same (or essentially the same) epitope specificity as the antibody AH8MAb, AH8L1, or AH12L3. 23. A binding agent comprising a variable region of an antibody AH8MAb, AH8L1, or AH12L3 (or a functional derivative thereof), for example, a Fab or Fab ′ fragment. 24. A dual acting heteroantibody comprising a first binding moiety that binds a first epitope and a second binding moiety that binds a second epitope, wherein the first and second epitopes are C-II _free Bifunctional heteroantibodies that are coupled epitopes. 25. An immunoassay kit comprising the antibody or the binding factor according to any one of claims 17 to 24. 26. A method (eg, a sandwich ELISA) for assaying for C-II _free in a biological sample (eg, a serum, urine, or SF sample), wherein the sample binds to a first epitope. Contacting a first antibody and a second antibody that binds to a second epitope, wherein the first and second epitopes are C-II _free coupled epitopes. 27. 27. The method of claim 26, wherein (a) the first antibody is immobilized; and / or (b) the second antibody is labeled; and / or (c) the antibody is And / or (d) said coupled epitope is defined as in any one of claims 5-8; and / or (e) said C-II _free. Is defined as in claim 4; and / or (f) the antibody is provided as part of an immunoassay kit as defined in claim 16. 28. An in vitro or ex vivo method of treatment, diagnosis, or prognosis, comprising assaying a biological sample for C-II _free according to the method of claim 26 or 27. 29. 17. A process for measuring the therapeutic activity of a drug, comprising: (a) providing a sample from a subject to be treated with the drug; and (b) any of claims 1-16. Assaying said sample using a kit according to any one of claims 17 to 24 or an antibody / binding agent according to any one of claims 17 to 24 or a method according to any one of claims 26 to 27. Wherein the amount of C-II _{free in said} sample assayed in step (b) is used as an indicator of therapeutic activity. 30. 30. A process for producing a therapeutic agent, comprising screening a library of candidate therapeutic agents for therapeutic activity using the process of claim 29. 31. 31. A process for producing a therapeutic agent, comprising: (a) selecting a chemical formula based on the structural characteristics of the therapeutic agent produced by the process of claim 30; and (b) step (a). Producing a therapeutic agent having the selected formula (eg, by synthesis or isolation). 32. A therapeutic agent obtainable (or obtained) by the process according to claim 30 or 31. 33. 32. The process according to any one of claims 29 to 31, wherein the therapeutic agent is an anti-arthritic agent or an MMP inhibitor. 34. A cell or cell line that expresses the antibody or binding agent according to any one of claims 17 to 24. 35. A process for producing an antibody that binds to a C-II _free coupled epitope, comprising: (a) degrading type II collagen in vitro to produce a plurality of fragments; (b) based on size (C) identifying a relatively large fragment; (d) screening the relatively large fragment for the presence of multiple epitopes; (e) having two or more epitopes Selecting a fragment; and (f) raising an antibody against the fragment selected in step (e). 36. An antibody that binds to a C-II _free coupled epitope obtainable (or obtained) by the process of claim 35. 37. An isolated type II collagen fragment having two or more epitopes for use in step (f) of the process of claim 35, wherein the fragment is, for example, a type II collagen α1 peptide. A fragment comprising (or consisting essentially of) an amino acid sequence from the N-terminal region (eg, from the N-terminal bridging region of the triple helix). 38. 38. The fragment of claim 37, wherein the sequence of 10-60 (eg, about 50) amino acids from the N-terminal region of the type II collagen α1 peptide (eg, from the N-terminal bridging region of the triple helix). A fragment comprising (or consisting essentially of).