MXPA01001692A - Assays for measurement of type ii collagen fragments in urine - Google Patents

Abstract

This invention provides novel methods for monitoring urine for type II collagen fragments.

Description

ESSAYS FOR THE MEASURE OF COLLAGEN FRAGMENTS OF TYPE HEN LAORINA REFERENCE TO A RELATED APPLICATION This application is a continuation in part of the patent application of E.U.A. Serial No. 09 / 184,658, filed on November 2, 1998, the entirety of which is incorporated herein by reference.

FIELD OF THE INVENTION This invention relates to methods for detecting fragments of type II collagen in biological media. More specifically, it deals with methods to quantify fragments of proteins found in urine that result from the breakdown of type II collagen.

BACKGROUND OF THE INVENTION The physiological replacement of articular cartilage represents a precise balance between synthesis and degradation. It is a characteristic of normal growth and development and maintenance of cartilage in adults.

A loss of net cartilage is a characteristic of arthritis. It is very associated with disabilities and a low quality of life. Cartilage destruction in rheumatoid arthritis and osteoarthritis is currently diagnosed based on the combination of clinical symptoms and radiological findings. Articular cartilage lesions occur early in the disease, long before it can be detected radiologically; lesions are detected radiologically only after extensive and probably irreversible loss of cartilage has occurred. Therefore, it is very important that physicians have biochemical markers for a diagnosis of cartilage lesions, so that therapy can be started soon, before extensive damage occurs. In addition, these markers can be used in the design of clinical trials in the selection of subjects for their participation, which are more likely to present a rapid progression. In addition, the effects of the treatment can be controlled as a function of time. The TUNE analysis can also provide information by which the effectiveness of the compound can be measured. Type II collagen constitutes the majority of the fibrillar skeleton of the cartilage matrix, just as type I collagen forms the fibrillar organization of the extracellular matrix of most other tissues, such as skin, bone, ligaments and tendons. The destruction of articular cartilage during arthritic diseases is due, in part, to the degradation of the extracellular matrix, which is composed mainly of fibrillar type II collagen and aggregation proteoglycans. In articular cartilage, type II collagen fibrils are responsible for structure and tensile strength, while proteoglycans provide the compressive stiffness required for joint and normal function. The precise mechanisms by which these connective tissue components are degraded are not fully understood. In mammals, an important mechanism applies to collagenases, MMP-1, MMP-8, MM-13 and MT1-MMP, which are a group of enzymes capable of breaking site-specific helical collagen (native). The three types of collagen are composed of a highly coiled triple helix, and in humans, collagenases break it extracellularly to produce chain fragments -a of% and% of length, which can be identified by a polyacrylamide gel electrophoresis. .

BRIEF DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides a method for controlling fragments of type II collagen in the urine, and said method comprises contacting said urine with a capture antibody, wherein said capture antibody binds in a similar manner. specific to the collagen type II fragments up to the substantial exclusion of any binding to the collagen fragments of type I or III, contacting said urine with a detection antibody, in which said detection antibody binds in a specific manner with collagenase fragments generated by collagenase, and detecting the amount of type II collagen fragments bound to said capture and detection antibodies.

In a preferred embodiment of the first aspect, the detection antibody is active against the sequences appearing in SEQ ID NO: 1 and 2. In another preferred embodiment of the first aspect, the detection antibody has a VH sequence that is at least 95%. % homologous with that appearing in SEQ ID NO: 32, and a VL sequence that is at least 95% homologous with that appearing in SEQ ID NO: 33. In another preferred embodiment of the first aspect, the detection antibody has the same CDRs as the VH sequence appearing in SEQ ID NO: 32, and the same CDRs as the sequence VL that appears in SEQ ID NO: 33. In another preferred embodiment of the first aspect, the capture antibody is active against the sequences appearing in SEQ ID NO: 3 and 4. In another preferred embodiment of the first aspect, the capture antibody has a sequence V which is at least 95% homologous to that appearing in SEQ ID NO: 48, and a sequence VL that is at least 95% homologous with the one that appears in SEQ ID NO: 49. In another preferred embodiment of the first aspect, the capture antibody has the same CDRs as the sequence V appearing in SEQ ID NO: 48, and the same CDRs as the sequence VL appearing in SEQ. ID NO: 49 Those skilled in the art will understand that the order in which the antibodies are contacted with the biological media can be reversed. In addition, in certain embodiments, this aspect of the invention comprises a method for controlling urine to detect fragments of type II collagens as described above, in which the contacting steps occur simultaneously. In another preferred embodiment of the first aspect, the steps of contacting the antibodies are produced sequentially, and after the contacting step of the capture antibody, and prior to the contacting step of the detection antibody, said capture antibody. it is immobilized on a magnetic material. In another preferred embodiment of the first aspect, the method described above includes other steps of contacting a series of control samples with said capture antibody, contacting said control samples with said detection antibody, and detecting the amount of fragments of type II collagen in said control samples that are linked to said capture and detection antibodies. In a second aspect of the present invention, there is provided an assay kit for controlling the urine to detect fragments of type II collagen, and said assay kit comprises a capture antibody, wherein said capture antibody binds in a specific to type II collagen fragments up to the substantial exclusion of any binding to type I or III collagen fragments, a detection antibody, in which said detection antibody binds specifically to the generated collagen fragments by collagenase, a container, and instructions describing a method for using said first antibody and said second antibody to control biological media to detect fragments of type II collagen. In a preferred embodiment of the second aspect, said detection antibody has a VH sequence that is at least 95% homologous to that appearing in SEQ ID NO: 32 and a VL sequence that is at least 95% homologous with that appearing in SEQ ID NO: 33, and said capture antibody has a VH sequence that is at least 95% homologous to that appearing in SEQ ID NO: 48, and a VL sequence that is at least 95% homologous with which it appears in SEQ ID NO: 49. In another preferred embodiment of the second aspect, said detection antibody has the same CDRs as the VH sequence appearing in SEQ ID NO: 32, and the same CDRs as the VL sequence appearing in the SEQ ID NO: 33, and said capture antibody has the same CDRs as the sequence VH that appears in SEQ ID NO: 48, and the same CDRs as the sequence V that appears in SEQ ID NO: 49. In another embodiment preferred of the second aspect, said test kit further comprises a positive control fluid comprising a urine control containing a known amount of type II collagen fragments, and a dilution fluid comprising the control urine to dilute the samples being tested with said test kit. In a third aspect of the present invention, a biodetector plate for detecting the presence of an immunological binding event is provided, wherein said plate comprises a first active antibody against the sequences appearing in SEQ ID NO: 1 or 2 , or a second active antibody to the sequences appearing in SEQ ID NO: 3 or 4. In a fourth aspect of the present invention, a method is provided for diagnosing a patient to detect a disease state associated with the degradation of the type II collagen, and said method comprises the step of detecting the presence of abnormally high amounts of type II collagen fragments in urine collected from said patient. In a fifth aspect of the present invention, there is provided a method for conducting a clinical trial to evaluate a drug that is believed to be useful for treating a disease state associated with the degradation of type II collagen, and said method comprises measuring the level of type II collagen fragments in urine collected in a series of patients, administering said drug to a first subset of said patients, and a placebo to a second subset of said patients, repeating said measurement step after administering said drug or said placebo, and determining whether said drug is reducing the amount of type II collagen fragments present in said urine from said first subset of patients, to a degree that is statistically significant compared to any reduction that is producing in said second subset of patients, in which a statistically significant reduction indicates that d The drug is useful for treating this disease state. In the present invention, a hybridoma cell line or E. coli culture that produces a monoclonal antibody that binds to peptides consisting essentially of the structure appearing in the sequence listing as SEQ ID NO: 1 or SEQ ID NO is useful. : 2, and the cell line has the identifying characteristics of ATCC HB-12436. Also useful in the present invention is a hybridoma cell line or E. coli culture that produces a monoclonal antibody that binds to peptides consisting essentially of the structure appearing in the sequence listing as SEQ ID NO: 3 or 4, and the cell line has the identifying characteristics of ATCC HB-12435.

DETAILED DESCRIPTION OF THE INVENTION Type II collagen is the structural protein that provides the articular cartilage with its resistance to stress and its resistance to shear. It also provides the structural basis for containing proteoglycans that impart sympathetic resistance, and thereby directly determines the osmotically pressurized cartilage shape. Therefore, the structural integrity of type II collagen is an important determinant of the physical properties and durability of articular cartilage. The progressive failure of the articular cartilage is one of the characteristics of arthritic diseases. When failure is based on changes in the structure of type II collagen, it will be advantageous to have a methodology to specifically measure the degradation of type II collagen. Fragments of type II collagen from the articular cartilage are released into the synovial fluid, then transported through the lymph, the blood, and released into the urine. Measurements of the released fragments could provide a method to control the degradation of type II collagen, to detect the onset of an arthritic disease and to measure the progress of the disease. In addition, it would also be useful to measure the effect of disease modifying therapy on the degradation of type II collagen during disease. A variety of methods have been used to control the degradation of collagen. In mammalian tissues, collagenase appears to be the extracellular enzyme that limits speed, involved in the extracellular degradation of type II collagen (1). Collagen collagenase fragmentation has been identified in% and% fragments as early as 1967 (2,3). At present, four mammalian collagenases (MMP-1, MMP-8, MMP-13 and MT1-MMP) involved in the initial breakage of type II collagen have been identified (4,5,29). Other enzymes are involved in the subsequent fragmentation of type II collagen. It is known that the lysosomal degradation of fibrillar collagens occurs in bone and liver (6,7). Since collagen is one of the few proteins characterized by a high content of hydroxyproline, the measurement of urinary hydroxyproline has been studied as a measure of collagen turnover (8). However, the method has not been useful to measure the degradation of type II collagen, since most of the signal is derived from type I and type II collagens, which are found in large amounts in the skin, bones and connective tissues. Therefore, it has no value in controlling type II collagen, since it only supplies an extremely small portion of the urinary secretion of hydroxyproline (8). Therefore, efforts to control collagen have focused on immunological detection methods. Antibodies can discriminate between collagen fragments unique to one type of collagen and one site of rupture, and can potentially control the specific mode of collagen degradation (9). Polyclonal and monoclonal antibodies against type I and III collagen and its fragments have been prepared, and assays have been prepared for the degradation products of type I and type III collagen, for example see Eyre (10). In fact, these tests are commonly used in clinics to control bone resorption. Relatively few methods have been indicated that use antibodies against the degradation products of type II collagen.

Polyclonal and monoclonal antibodies against type II collagen have been prepared (9,11-13). These antibodies have been used for the detection of intact type II collagen, rather than for the quantitative determination of collagen fragments. However, Eyre (14) has prepared monoclonal antibodies against fragments of type II collagen that contain the crosslinking residues. He has developed an assay for the degradation of type II collagen based on the cross-linking fragment containing a specific sequence of type II collagen similar to that of his filed patent (10). Dodge and Poole have prepared polyclonal antibodies against denatured type II collagen, which were not reactive with native type II collagens or other collagens (15,16). The epitope was sequenced, and then Hollander and Poole (17,18,19) prepared a competitive antibody assay against fragments of type II collagen having the sequences appearing in the sequence listing as SEQ ID NO: 12 and 13 , using a monoclonal antibody. Hollander and Croucher (20) also performed a capture ELISA using antibodies directed against the peptides that appear in the sequence listing as SEQ ID NO: 67, 68 or 69. Billinghurst et al. (21) have prepared a polyclonal antibody against the collagen type II breakage site neoepitope (which has the sequence indicated in the sequence listing as SEQ ID NO: 2), and have prepared a competitive type II collagen assay . Srinivas, Barrach and Chichester (22-24) have prepared multiple monoclonal antibodies for a type II collagen assay using the cyanogen bromide fragments of type II collagen as antigen. Although epitopes reactive with the antibody have not been identified (25), they are also capable of testing type II collagen. A capture ELISA is a reliable method to obtain a high specificity, since it is based on two antibodies that recognize in a coordinated way two different epitopes of amino acids in the same molecule. Since the antibody binding site contains only six amino acids, the two capture assay antibodies can recognize sequences as small as 15 to 20 amino acids. Competitive assays use a single antibody, and therefore allow the measurement of polypeptides as small as 6-8 amino acids, but lack the specificity of dual antibody measurement. In addition, competitive assays lack the sensitivity of capture ELISAs, which often have a detection limit 100-10,000 times lower. Therefore, capture ELISAs provide a preferred method for measuring protein metabolic fragments. For example, a capture ELISA has been used to measure fragments of degradation of the structural protein elastin in blood (26). A capture ELISA has been used to measure the biologically active peptide of 21 amino acids endothelin (27). A pair of capture ELISAs have been used to measure the different metabolic fragments of the glucagon peptide of 28 amino acids (28). Based on these results, a peptide fragment with a minimum of 22 amino acids was selected, which could be used to construct a capture ELISA to measure the collagenase-dependent metabolism of type II collagen. This invention provides two types of assays for type II collagen metabolism. Both assays are based on an antibody (polyclonal, monoclonal or an antibody generated by genetic engineering) against a defined sequence of type II collagen, against which antibodies have not previously been prepared. The sequence is rich in acidic residues, that is, the sequence that appears in the sequence listing as SEQ IND NO: 3 in which a deletion of 1 residue has been made at the C-terminal end. Since no aspartyl endopeptidase or extracellular glutamyl has yet been described in mammals, it is expected that collagen fragments rich in acidic residues will survive the subsequent metabolism. Therefore, these fragments must be measurable in body fluids. An antibody against these residues or a fragment of collagen containing these residues would provide a general method for the detection of type II fragments in body fluids, independent of the method of generating the collagen metabolite. This invention provides monoclonal antibody 5109 and variants of 5109 genetically engineered which are specific for the type II collagen sequence, and which bind to the collagen fragments containing the sequence. The first trial is a general method to evaluate the degradation of type II collagen. This assay provides a general competitive method for quantifying the amount of sequence that appears in the sequence listing as SEQ ID NO: 3, in which a deletion of 1 residue has been made at the C-terminal end, and its closely related congeners. For the second assay, another antibody was prepared against the sequence appearing in the sequence listing as SEQ ID NO: 14. There is a free C-terminal carboxyl group in the glycine (residue 9 of SEQ IND NO: 14). This sequence is obtained when the collagenase breaks type II collagen, and therefore it is classified as a neoepitope, that is, it is not present in the native sequence (the sequence that appears in the sequence listing as SEQ ID NO: 15, continues GPOGPQG / LAG- and collagenase breaks in the vertical bar), but arises when collagenase breaks down collagen. Polyclonal antibodies against this sequence have previously been indicated (22). The monoclonal antibody 9A4 and its genetically engineered derivatives react with the sequence of the neoepitope that appears in the sequence listing as SEQ ID NO: 2, but do not react with type I collagen or type II without breaking. Although the neoepitope sequence is exclusive of type II collagen when it is broken by coiagenase, a homologous sequence is generated and has a weak cross-reaction in type I collagen when it is broken by collagenase (SEQ ID NO: 16 sequence listing). This is also true for lll-type collagen; generates the sequence that appears in the sequence listing as SEQ ID NO: 2 when it is broken by collagenase. Thus, the neoepitope antibody lacks total specificity for type II collagen, nor could it selectively detect the breakdown of collagen type II by collagenase if used alone. However, when the 5109 antibody and the 9A4 antibody are combined in a sandwich assay, both antibodies can selectively detect the type II collagen metabolites generated by the collagenase. In addition, an advantage provided by the sandwich assay in the present invention is a detection limit 100 times lower, compared to a simple competitive assay based only on 9A4. Therefore, the sandwich assay format with the antibodies described in this invention provides a unique method for controlling the metabolism of collagen type II by collagenase under normal and pathological conditions, which have not been previously described. On its own, the 9A4 antibody is a new monoclonal antibody that can be used for the detection of fragments of collagen type I, or type II, or type III, broken by collagenase, as long as it is not necessary to distinguish the type of collagenase. Collagen BIBLIOGRAPHIC REFERENCES 1. Harris et al., N. Engl. J. Med., 291: 557-563, 605-609, 652-661 (1974). 2. Nagai et al., Biochemistry, 5: 3123-3130 (1966). 3. Sakai et al., Biochemistry, 6: 518-528 (1967). 4. Pendas et al., Genomics, 26: 615-618 (1995). 5. Mitchell et al., J. Clin. Invest., 97: 761-768 (1996). 6. Ac.ewicz et al., FEBS Lett., 269: 189-193 (1990). J. van Noorden et al., Bioc. Biop. Res. Comm., 178: 178-184 (1991). 8. Kivirikko, Int. Rev. Connect. Tissue Res., 5: 93-163 (1970). 9. Tirnpl, Methods Enzymol., 82: 472-498 (1982). 10. Eyre, US Patent 5,320,970 (1994). 11. Holmdahl et al., Immunology, 61: 369-374 (1987). 12. Punjabi et al., J. Immunol., 141: 3819-3822 (1988). 13. Jasin et al., J. Clin. Invest., 87: 1531-1536 (1991). 14. Noriund et al., Trans. Orthoped. Res. Assoc, 22: 313 (1997). 15. Dodge et al., J. Clin. Invest., 83: 647-661 (1989). 16. Dodge et al., Matrix, 11: 330-338 (1991). 17. 17. Poole, PCT publication WO 94/14070 (1994). 18. Hollander et al., J. Clin. Invest. 93: 1722-1732 (1994). 19. Hollander et al., J. Clin. Invest. 96: 2859-2869 (1995). 20. Hollander et al., PCT publication WO 98/3523520 (1998). 21. Billinghurst et al., J. Clin. Invest., 99: 1534-1545 (1997). 22. 22. Srinivas et al., J. Immunol. Meth., 159: 53-62 (1993). 23. Srinivas et al., Agents Actions, 41: 193-199 (1994). 24. Srinivas et al., Immunol. Invest., 23: 85-98 (1994) 25. Chichester et al., Pharm. Pharmacol., 48: 694-698 [lagoon] 26 Baydanoff et al., Atherosclerosis, 66: 163-168 (1987). 27. Hamaoki et al., Hybrídoma, 9: 63-69 (1990).

Definitions Immunoglobulin (Ig): A natural tetrameric protein composed of two light chains of approximately 23 kD and two heavy chains of approximately 53-70 kD, depending on the amino acid sequence and the degree of glycosylation. Multimers of the tetrameric protein (IgM and IgA) are also formed. There are two classes of light chains, kappa (K) and lambda (?), And several classes of heavy chains, gamma (?), Mu (μ), alpha (a), delta (d) and epsilon (e). There are also subclasses. Each chain, light or heavy, is made up of two parts. The first part, which starts from the N-terminal end of any of the chains, is called the variable domain. The C-terminal half of the light chain is called the constant region of the light chain, and it is the main determinant whether the light chain is of type K or type?. The constant region of the heavy chain comprises approximately three fourths C-terminal of the heavy chain, and determines the class of immunoglobulin molecule (IgGi, IgM, etc.), ie, a heavy chain? corresponds to an IgG, and a heavy chain μ corresponds to an IgM, etc. Vj ^ and VH: The amino acid sequence of the variable domain of the light chain (V) and the variable domain of the heavy chain (VH) together determine the binding specificity and the binding constant of the immunoglobulin molecule. The variable domain comprises approximately half the length of the light chain, and about a quarter of the length of the heavy chain, and for both chains it begins at the N-terminus of the chain. The variable regions each contain three hypervariable segments, known as the regions determining complementarity or CDR. CDR and FR: Each variable domain, VL or VH, is formed by three CDRs: CDR1, CDR2 and CDR3. The intermediate sequence segments before, between and after the CDRs are called work frame segments (FR). Each V and VH is formed by four FR segments: FR1, FR2, FR3 and FR4. V? __ _ \ 6t: The VL domain is K or?, Depending on the constant region (C? Or C?) Used during the productive rearrangement of the light chain (VJCK OR VJC?) - Antibody: Antibodies are molecules of Specific immunoglobulin produced by the B cells of the immunological system in response to exposures to proteins, glycoproteins, viral cells, chemicals coupled to vehicles and other substances. An antibody is simply an immunoglobulin molecule whose binding partner is known. The substance to which the antibody binds is called an antigen. The binding of these antibodies to their antigen is very refined, and the multitude of specificities capable of being generated by changes in the amino acid sequence in the variable domains of light and heavy chains is remarkable. Polyclonal antibody: Normal immunization leads to a wide variety of antibodies against the same antigen. Although each B lymphocyte normally produces an immunoglobulin molecule with a defined amino acid sequence, in an immune response many B lymphocytes are stimulated to produce immunoglobulin molecules that react with the antigen, i.e., antibodies. These different antibodies are characterized by different amino acid sequences in the variable regions of the immunoglobulin molecule, which result in differences in precise specificity and binding affinity. These antibodies are called polyclonal antibodies, to emphasize the diversity of binding specificities and binding constants that arise from the diversity of amino acid sequences found in the different immunoglobulin molecules used in the immune response. Monoclonal Antibody (mAb): A B lymphocyte that produces a single antibody molecule can hybridize to an immortal B-cell line, i.e., a myeloma, to derive an immortal cell line that produces antibodies, i.e., a hybridoma. Hybrids formed in this way are segregated into unique genetic strains by selection, dilution, subcloning and recultivation, and each strain thus represents a single genetic line. It produces a single antibody with an exclusive sequence. The antibody produced by said cell line is called "monoclonal antibody" or mAb, making reference to its pure genetic lineage, and differentiating it from the polyclonal antibody, produced by a mixed genetic environment, that is, by multiple B cells. Because a mAb It is a pure chemical reagent, produces consistent and consistent results in immunological assays. In addition, because the mAb is produced by an immortal cell line, the reagent supply is not limiting. For these reasons, a mAb is more preferred than polyclonal antibodies for diagnostic purposes. Antibody produced by genetic engineering: Since the binding specificity of an antibody resides in the variable regions of light and heavy chains, antibodies can be produced by genetic engineering so that constant regions change or are eliminated, and if it is carried out in a In a suitable manner, an antibody molecule with different properties and molecular weight can be produced, but with the same or very similar antigen-binding properties. For example, the VL and VH genes can be cloned and assembled (or VH and VL) with an appropriate linker between them. This new genetically engineered molecule is called the single-chain antibody (abbreviated scFv), and typically has a molecular weight of 25-28 kD, depending on the design of the linker and the addition of other sequences to aid purification , stability, transport, detection, etc. Single-chain antibody multimers can also be produced by the appropriate use of connectors, in which the order of each VHA L pair can vary. In addition, some changes in the amino acid sequence of the VH and VL region may be made while maintaining the desirable antigen binding properties. It can be understood that an infinite variety of antibodies produced by genetic engineering can be derived from the original antibody sequence, while maintaining the specificity of antigen binding, but that they are designed to meet specific requirements. Other examples of antibodies produced by genetic engineering include, but are not limited to Fab, F (ab ') 2, chimeric antibodies, humanized antibodies, etc. For a report, see Winter and Milstein, Nature, 349: 243-299 (1991). Bispecific Antibody: Normally, an IgG antibody has two identical light chains and two identical heavy chains. Thus, there are two identical antibody binding sites in the immunoglobulin molecule. In contrast, a bispecific antibody is a single immunoglobulin molecule that has two different specificities. It can be formed by fusion of two hybridoma cell lines that produce monoclonal antibodies, each hybridoma having a different antigen specificity, and the selection of a cell line (one quadroma) that produces an antibody whose composition is a tetramer composed of a light chain and a heavy chain from each hybridoma fusion partner. The antibody produced by the quadroma has only one light and one heavy chain of each parent specificity, and has a binding site for each pair of heavy / light chains and is bispecific, ie it has two binding sites of different specificity. A bispecific antibody can also be produced by genetic engineering. It may comprise the V-connector-Vp of an antibody linked through another linker to a VL-Vp-linker of another antibody molecule. The order of V and VH can be altered, but the final result is a bispecific antibody. Epitope: Depending on the size, structure and conformation of the antigen, an antibody can bind only a small part of the total structure. The part of the antigen molecule to which the antibody binds is called its epitope. Different antibodies can be mapped for different epitopes on the same antigen. Neoepitope: The antigen can have an epitope that is hidden, so that it can not bind to a specific antibody. However, a conformational change in the antigen can cause the appearance of the epitope by unfolding or discovering part of the surface of the molecule. This now allows the antibody to bind to the epitope. In another aspect, the action of an enzyme on the antigen can cause the birth of a new epitope to which the antibody can bind. For example, after breakage by a proteolytic enzyme, new N-terminal and C-terminal sequences are generated. Because the epitope is not observed in the parent molecule and that after some change in the parent molecule, the epitope is revealed and can now bind to an antibody, it is called a neopitope.

TUNE: English abbreviation of the phrase "neoepitope of type II collagen". This expression is used to refer to the specific neoepitope of collagen recognized by the 9A4 antibody. Biological medium: Can be defined as any biological fluid that can contain the antigen, and that may be of interest for the testing of this procedure. These include, blood, synovial fluid, urine, feces, sperm, saliva, spinal fluid, bronchiolar lavage fluid, lymph, vitreous humor of the eye, tissue extracts, tissue culture supernatants, cartilage extracts, etc. Biological media are not necessarily limited to human samples, but can also be obtained from a similar variety of animal media (media from mouse, rat, hamster, guinea pig, dog and bovine) have been tested in a manner similar to the previous examples . Immunoassay: An assay for a substance (a biological complex such as a protein, or a simple chemical compound) based on the use of the binding properties of the antibody to recognize a substance, which may be a particular molecule or a series of homologous molecules. The assay may involve one or more antibodies. Direct assay: The antibody binds directly to an antigen as in a biological specimen (cells, tissues, histological sections, etc.), or to an antigen absorbed or chemically coupled to a solid surface. The antibody itself is often labeled to allow determination of the amount of antibody bound to the antigen.

Alternatively, the antibody (now called primary antibody) is detected with a labeled secondary antibody, which demonstrates that the binding of the primary antibody has occurred. Competitive assay: An assay based on the binding properties of a single antibody molecule. Typically, a labeled antigen is used to compete with an unknown antigen, and the amount of unknown antigen is determined in terms of the amount of labeled antigen displaced by the unknown antigen. The label can be radioactive, optical, enzymatic, fluorescent polarizing, fluorescent by extinction or another marker. The antibody can be monospecific or bispecific. Sandwich sandwich: It is a double antibody test, in which both antibodies bind to the antigen, forming a trimeric or sandwich immune complex, which contains the two antibodies with the antigen between them, an antibody is used to place the immune complex in the camera or detection surface. This antibody is called capture antibody. The other antibody has a marker that allows the detection of the immune complex. It is called detection antibody. If the immunological complex is not formed (the antigen is not present), then the capture antibody is unable to carry the detection antibody to the detector. If the antigen is present, then an immunological complex will be formed and the capture antibody will bind with the detection antibody, so that the amount of detection antibody in the immune complex is quantitatively related to the amount of antigen present. The trial can have several formats. For example, the capture antibody can be chemically coupled to a solid surface, or it can be non-specifically absorbed onto a surface, it can be linked by biotinylation to a molecule of the avidin type (for example avidin, streptavidin, neutravidin, etc.) or to an avidin-coated surface, or it may be coupled to spheres or magnetic particles, all these means being to place the immunological complex in the measuring device. The detection antibody can be radiolabelled, or it can have a variety of possible enzyme amplification systems, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), urease, etc., when presented in an ELISA format (enzyme-linked immunosorbent assay). You can use an electrochemical, fluorescent detection method or other method to determine the amount of detection antibody in immune complexes. It can immediately be seen that many examples can be derived, in which the two antibodies are paired in a sandwich assay using a variety of methods to capture the immunological complex in a detection device, and a variety of detection systems to measure the amount of immune complex.

Molecular biology techniques: use the nucleotide sequences of the V and V H coding regions are supplied for the antibodies of the present invention, an expert can produce in vitro a complete gene encoding the VH and VL regions and an antibody completely functional. It can be produced as an immunoglobulin molecule of any given kind in the constant regions of the added heavy chain and light chain, or it can be produced as a scFv with V and VH attached by a linker with added labels if necessary. The constructed gene can be modified by conventional recombinant techniques, for example, to deliver an inserted gene into a plasmid capable of expression. After this, the plasmids can be expressed in host cells, in which the host cells can be bacteria such as E. coli or a Bacillus spice, yeast cells such as Pichia pastoris, or in mammalian cell lines such as Sp2 / 0, Ag8. or CHO cells. Homology: In the present application, when reference is made to the homology of an amino acid or a nucleic acid, BLASTP, BLASTN and FASTA have been used (Atschul et al., J. Mol. Biol., 215: 403-410 (1990 )) to calculate the homology, unless otherwise indicated. The BLAST X program is available to the public at NCBI and other sources (BLAST Manual, Altschul et al., NCBI NLM NIH Bethesda, MD 20894, and Altschul et al., Id.).

Abbreviations When abbreviated nucleic acids, amino acids, peptides, protective groups, active groups and similar residues, are abbreviated according to the IUPACIUB (Commission on Biological Nomenclature) or practice in related fields. The following are examples.

Conventional abbreviations HPLC: high resolution liquid chromatography SDS-PAGE: polyacrylamide-sodium dodecyl sulfate gel electrophoresis PCR: Oligo polymerase chain reaction: oligonucleotide TA: room temperature, approximately 22 ° C EDTA reagents: ethylenediaminetetraacetic acid SDS: sodium dodecylsulfate TW-20: Tween-20 NFDM: skimmed milk powder DPBS: Dulo phosphate buffered saline Bt: biotinylated HAT: hypoxanthine-containing medium, aminopterin, thymidine HT: medium containing hypoxanthine, thymidine HRP: horseradish peroxidase Chains or molecules of the immunoglobulin type VH or VH: variable region of the heavy chain VL OR VL: variable region of the light chain ScFv: chain antibody containing a V and a VH Nucleic acids RNA: ribonucleic acid DNA: deoxyribonucleic acid cDNA: complementary DNA mRNA: messenger RNA Bases of nucleic acids Amino acids-single-letter codes: three-letter codes: full names EXAMPLES EXAMPLE 1 Generation and characterization of monoclonal antibody 9A4 Balb / c mice (Jackson Laboratories, Bar Harbor, ME) were immunized initially with the peptide listed in the sequence listing as SEQ ID NO: 17 (Anaspec, San Jose, CA) covalently linked to the maleimide KLH (Pierce Chemical, Rockford, IL) and administered in complete Freund's adjuvant (DIFCO Detroit, Ml). The mice received a booster immunization every month for approximately 5 months using incomplete Freund's adjuvant (DIFCO, Detroit, Ml) until a value of 1: 100,000 was reached. The mice received an intravenous booster immunization 10 days before fusion. Splenocytes were harvested and fused with a non-secreting cell line Ig derived from P3X63Ag8.653 (American Type Culture Collection, Bethesdam MD) using 50% PEG-1450 (ATCC). They were plated at 106 cells / well in 96-well microtiter plates in HAT medium (Sigma, St. Louis, MO) with 15% fetal calf serum (Hyclone, Provo, Utah). Ten days later, the wells were investigated with a primary ELISA. For the identification of positive antibody producing wells, 10 ng / ml biotinylated peptide (Bt-AEGPPGPQG) [biotinylated in residue 1 of SEQ ID NO: 14] was added to the streptavidin-coated plates (10 μg / ml) (Pierce Chemical), and 2 μl of each Hybridoma supernatant were added to 100 μl of DPBS (Gibco, Grand Island, NY) with TW-20 ai 0.05% (Sigma). Positive wells by ELISA were detected by rabbit anti-HRP-lgG (Jackson Immuno Research, West Grove, PA). The positive wells by ELISA underwent a second round of selection with the BIAcore ™ system. We searched for wells that produced antibodies with slower exit rates in the BIACore ™ system, as determined using the BIAevaluation ™ version 2.1 program (Pharmacia Biosensor, Piscataway, NJ). Streptavidin (Pierce Chemical) was conjugated at 100 μg / ml using the Pharmacia amine coupling assay kit (Pharmacia Bionsensor) to carboxylated dextran-coated biodetector plates (Pharmacias Biosensor) at pH 4.0, using a flow rate of 5 μl / minute for 35 minutes. Typically, RU 2000 was added. The peptide (100 ng / ml) biotinylated in residue 1 of SEQ ID NO: 14, as indicated in the sequence listing, was passed over the streptavidin plate at a rate of 100 μl / minute for 10 seconds. The candidate supernatants containing antibodies were passaged on the plate (2 μl / minute for 30 seconds), and the amount of antibody added was noted. The buffer was changed to HBS (HEPES-buffered saline, Pharmacia Biosensor), and the dissociation rate was noted for the next 80 seconds. The plate was cleansed with 0.1 N HCl for 30 seconds between each assay, to remove residual antibodies and to eliminate any non-specific binding. The output speeds were determined using a BIAevaluation ™ kinetic analysis program version 2.1. The clones with the lowest exit velocity were selected for further analysis. These clones included 9A4, 11 F2 and 3H10. The subsequent characterization of these clones was carried out as follows: four preparations consisting of type I collagen, type I collagen broken by collagenase, type II collagen, and type II collagen broken down by collagenase were each coupled to a different flow cell in a BIA 2000 instrument. They were conjugated using the Pharmacia amine coupling assay kit (Pharmacia Biosensor) to carboxylated dextran-coated biodetector plates (Pharmacia Biosensor) at pH 4.0 using a flow rate of 5 μl / minute for 35 hours. minutes The four flow cells added 8000, 7000, 4000 and 4000 RU, respectively. The cells were washed with 0.1 N HCl to clean them of any uncoupled material, and to clean them of any residual antibody between the assays. All antibody preparations were purified by protein G chromatography (Pharmacia Biotechnology, Piscataway, NJ) and assayed at 10 μg / ml. The total union to each of the surfaces was recorded. The 9A4 antibody was selected because it showed a selective binding to type II collagen broken down by collagenase (type 1 = 11 RU; type II = 280 RU) and did not present a significant union with collagen without breaking (type I = 3 RU, type II = 6 RU). After three rounds of subcloning by limiting dilution in HT medium (Sigma) with 5% fetal calf serum (Hyclone), a stable 9A4 monoclonal hybridoma was obtained. It has been deposited in the American Type Culture Collection as ATCC-HB-12436.

EXAMPLE 2 Generation and characterization of monoclonal antibody 5109 Balb / c mice were immunized initially with the peptide listed in the sequence listing as SEQ ID NO: 18 (Anaspec, San Jose, CA) covalently bound to the maleimide KLH (Pierce Chemical) and administered in complete Freund's adjuvant (DIFCO) ). The mice received a booster immunization each month for approximately 5 months using incomplete Freund's adjuvant (DIFCO) until a value of 1: 100,000 was reached. The mice received an intravenous booster immunization 10 days before fusion. Splenocytes were collected and fused with a non-secreting cell line Ig derived from P3X63Ag8.653 (ATCC) using 50% PEG-1450 (ATCC). They were plated at 106 cells / well in 96-well microtiter plates in HAT medium (Sigma) with 15% fetal calf serum (Hyclone). Ten days later, the wells were investigated by ELISA. For the identification of the positive antibody-producing wells, 10 ng / ml biotinylated peptide (biotinylated in residue 1 of SEQ ID NO: 19, as shown in the sequence listing) was added to streptavidin-coated plates (10 μg / ml). ml) (Pierce Chemical) and 2 μl of each hybridoma supernatant was added to 100 μl of DPBS with 0.05% TW-20 (Sigma). Positive wells by ELISA were detected by rabbit anti-HRP-IgG (Jackson ImmunoResearch). The positive wells underwent a second round of selection with the BIAcore ™ system for those with antibodies with slower exit velocities. Streptavidin (Pierce Chemical) was conjugated at 100 μg / ml using the Pharmacia amine coupling assay kit (Pharmacia Biosensor) to carboxylated dextran-coated biodetector plates (Pharmacia Biosensor) at pH 4.0, using a flow rate of 5 μl / minute for 35 minutes. Typically, 2000 RU was added. The peptide (100 ng / ml) biotinylated in residue 1 of SEQ ID NO: 19, as indicated in the sequence listing, was passed over the streptavidin plate at a flow rate of 100 μl / minute for 10 seconds. Supernatants containing antibodies were passed over the plate (2 μl / minute for 30 seconds), and the amount of antibody added was noted. The buffer was changed to HBS (Pharmacia Biosensor), and the dissociation rate was noted for the next 80 seconds. The plate was cleaned with 0.1 N HCl for 30 seconds between each test, to remove the antibodies and to eliminate any non-specific binding. The output speeds were determined using a BIAevaluation ™ version 2.1 program. The clones with the lowest exit velocity were searched. The 5109 mAb was selected for further analysis.

Four preparations consisting of type I collagen, type I collagen broken by collagenase, type II collagen, and type II collagen broken by collagenase were each attached to a different channel of a four channel BiAcore ™ system. They were conjugated using the Pharmacia amino-coupling assay kit (Pharmacia Biosensor) to carboxylated dextran-coated biodetector plates (Pharmacia Biosensor) at pH 4.0 using a flow rate of 5 μl / minute for 35 minutes. The four flow cells added 8000, 7000, 4000 and 4000 RU, respectively. The cells were washed with HCl, 0.1 N to clean them of any material not coupled, and to clean them of any specific material between the tests. All antibody preparations were purified by protein G chromatography (Pharmacia Biosensor) and assayed at 10 μg / ml. The total union was recorded to each of the four surfaces. Antibody 5109 was selected because it showed a selective binding to collagen broken by collagenase (type I broken = 23 RU, type II broken = 173 RU) and showed no significant binding to collagen without breaking (type I = 23 RU; = 15 RU). After nine rounds of subcloning by limiting dilution in HT medium (Sigma) with 5% fetal calf serum (Hyclone), a stable monoclonal hybridoma 5109 was obtained. It has been deposited in the American Type Culture Collection as ATCC-HB-12435.

EXAMPLE 3 Description of sandwich sandwich using 9A4 as the capture antibody and monoclonal antibody 5109 as the detection antibody Monoclonal antibody 9A4 (capture antibody) was added to Nunc Maxisorp ™ 96 well plates (VWR, Boston, MA) with 9A4 at 10 μg / ml in 0.05 M sodium borate buffer, pH 8.5, using 100 μl / well (except in control wells numbered 4, 5 and 6, see table 1) and incubated for 18-48 hours at 4 ° C. The plate was washed three times with DPBS containing 0.05% TW-20 (Sigma) (DPBS / TW-20); 200 μl / well was used. The wells in the plates were blocked with freshly prepared powdered milk (NFDM) dissolved in fresh DPBS (NFDM DPBS), that is kept on ice for no more than the day of use, using 100 μl / well incubated for 1 hour at RT. The block solution was removed, and the wells were rinsed once with 200 μl of DPBS / TW-20. Peptide 130 was diluted with 0.1% NFDM DPBS to the concentrations shown in Table 1. Peptide 130 has the sequence that appears in the sequence listing as SEQ ID NO: 20, and was synthesized and purified by Anaspec Inc. ( San José, CA).

Dilutions of peptide 130 (SEQ ID NO: 20), specimens in the appropriate solutions, and controls were placed in the specified wells of the microtiter plate, as shown in Table 1.

TABLE 1 Summary of antibody coating program and microtiter plate TABLE 2 Additions to control wells The wells were washed three times with 200 μl / well of DPBS TW-20. Biotin conjugated 5109 (Bt-5109) mAb was added to all wells containing peptide 130 (SEQ ID NO: 20), all sample wells, and all control wells except 1, 2 and 5. Bt was added. -5109 (100 μl / well) at 1 μg / ml in 0.1% NFDM DPBS to each well and the plate was incubated for 40 minutes at 37 ° C. Note: mAb 5109 was biotinylated using 37 μg of biotin-N-hydroxysuccinamide (Pierce Chemical) per mg of mAb 5109 for 2 hours and then dialyzed overnight using a dialysis module with an exclusion limit of 10 kD (Pierce). Chemical). The wells were washed three times with 200 μl / well of DPBS / TW-20. Anti-mouse biotin monoclonal antibody conjugated to HRP (Jackson ImmunoResearch) was diluted 1/5000 in 0.1% NFDM DPBS and added to 100 μl / well in all wells and incubated for 30 minutes at RT. The wells were washed three times with 200 μl / well of DPBS / TW-20. 100 μl / well of 1-step Turbo (3, 3 ', 5, 5'-tetramethylbenzidine ready for use, Pierce Chemical) was added to each well and incubated at RT for about 10 minutes. The color development was stopped with H2SO4 2 N. The results were read on a spectrophotometer at 450 nm.

TABLE 3 Data of the standard curve for sandwich sandwich with 9A4 capture / Bt- 5109 detection From the concentrations of peptide 130 and the reading of the resulting optical density at 450 nm (Table 3), a standard curve was constructed (Figure 1). Other suitable peptides or collagen fragments can be used to prepare a standard curve analogous to Figure 1. Units were expressed "in terms of standard molar equivalents .. In this case, the units were nM equivalent of peptide 130 (SEQ ID NO: twenty).

In the linear portion of the curve, a regression line was used to fit the data. In the given case, the standard curve was linear between 0J35 mM and 0.46 nM when the concentrations are given in the logarithmic scale (as in the example, figure 1) and the regression curve is obtained using only the linear portion of the curve. For samples that are left out of the linear portion, the concentrations can be read off the chart or the samples can be diluted to be within the standard portion of the curve, or they can be below the detection limit. The regression between log (nM) and DO450 produces a slope of 0.029 OD450 / log (nM) and an intersection of 0.024 OD450. When assaying unknown samples, the calibration curve can be used to determine the concentration of type II collagen fragments generated by collagenase from the optical density of the sample. The following equation can be used: Log (concentration) = (OD450 of the sample-intersection) / slope Sample: In the given case, an unknown sample of synovial fluid had an OD450 of 0.229. Therefore: Log (concentration) = (DO450 of the sample-0.024) /0.029=-0.949 Taking the anti-logarithm, the concentration of the fragment in the synovial fluid = 0.112 nM.

EXAMPLE 4 Description of sandwich sandwich using monoclonal antibody 5109 as capture antibody and 9A4 as detection antibody Monoclonal antibody 5109 (capture antibody) was added to Nunc Maxisorp ™ 96-well plates (VWR, Boston, MA) with 5109 at 10 μg / ml in 0.05 M sodium borate buffer, pH 8.5, using 10 μl / well (except in the control wells numbered 4, 5 and 6, see table 4) and incubated for 18-48 hours at 4 ° C. The plate was washed three times with 200 μl / well of DPBS / TW-20. The wells in the plates were blocked with 100 μl / well 1% NFDM dissolved in fresh DPBS, and incubated for 1 hour at room temperature. The block solution was removed, and the wells were rinsed once with 200 μl of DPBS / TW-20. Peptide 130 (SEQ ID NO: 20) was diluted with 0.1% NFDM DPBS to the concentrations shown in Table 4. The dilutions of peptide 130 (SEQ ID NO: 20), the unknown specimens in the appropriate solutions, and the controls were placed in the specified wells of the micro-score plate, as shown in Table 4.

TABLE 4 Summary of antibody coating program and microtiter plate TABLE 5 Additions to control wells The wells were washed three times with 200 μl / well of DPBS / TW-20. Monoclonal antibody 9A4 conjugated with biotin (Bt-9A4) was added to all wells containing peptide 130 (SEQ ID NO: 20), all sample wells, and all control wells except 1, 2 and 5. added 100 μl / well of Bt-9A4 at 1 μg / ml in 0.1% NFDM DPBS to each well and the plate was incubated for 40 minutes at 37 ° C. Note: 9A4 was biotinylated using 37 μg of biotin-N-hydroxysuccinamide (Pierce Chemical) per mg of monoclonal antibody 9A4 for 2 hours and then dialyzed overnight using a dialysis module with an exclusion limit of 10 kD (Pierce Chemical). The wells were washed three times with 200 μl / well of DPBS / TW-20. Anti-mouse biotin monoclonal antibody conjugated to HRP (Jackson ImmunoResearch) was diluted 1/5000 in NFMD 0.1% DPBS and added to 100 μl / well in all wells and incubated for 30 minutes at RT. The wells were washed three times with 200 μl / well of DPBS / TW-20. 100 μl / well of 1-step Turbo ™ (ready-to-use 3,3 \ 5,5'-tetramethylbenzidine, Pierce Chemical) was added to each well and incubated at RT for approximately 10 minutes. The growth of the cotor was stopped with H2SO 2 N. The results were read on a spectrophotometer at 450 nm.

TABLE 6 Data from the standard curve for the sandwich assay with 5109 capture / Bt-9A4 detection From the concentrations of peptide 130 (SEQ ID NO: 20) and the reading of the resulting optical density at 450 nm, a standard curve was constructed. Again, other suitable peptides or collagen fragments can be used to prepare a standard curve. The units needed were expressed in terms of pattern equivalents. In this case, the appropriate units were nM equivalents of peptide 130 (SEQ ID NO: 20). In the linear portion of the curve, a regression line was used to fit the data. In the given case, the standard curve was linear between 0.3125 nM and 0.0195 nM when the concentrations are given in the logarithmic scale (as in the example, figure) and the regression curve is obtained using only the linear portion of the curve. For samples that are left out of the linear portion, the concentrations can be read off the chart or the samples can be diluted to be within the standard portion of the curve, or the concentration of the collagen fragments in the sample can be below the detection limit. The regression between log (nM) and DO450 nm produces a slope of 0.42 OD450 / log (nM) and an intersection of 0.70 OD450 nm. When testing samples, the calibration curve can be used to determine the concentration of collagen fragments from the optical density of the sample.Sample: In the given case, an unknown sample of human urine from an arthritic patient had an OD450 of 0.124. Log (concentration) = (OD450 in the -0.70 way) / .42 = -1.36 Taking the anti-logarithm, the concentration of the fragment in the urine = 0.44 nM.

In another case, the standard curve produces a curve of 0.249 and an intersection of 0.638. An unknown sample of human osteoarthritis plasma had an OD450 nm of 0.172. The osteoarthritis plasma sample had a fragment concentration of 68 pM.

EXAMPLE 5 Antibody 5109 can be used directly to measure the amounts of the type II collagen fragment in a competition assay In an adaptation of the concentration analysis (BlAapplications Handbook, Pharmacia Biosensor, June 1994 edition, pp. 6-2 to 6-9), streptavidin (Pierce Chemical, Rockford, IL) was conjugated at 100 μg / ml with the test kit of Pharmacia amine coupling (Pharmacia Biosensor) to carboxylated dextran-coated biodetector plates (Pharmacia Biosensor) at pH 4.0 using a flow rate of 5 μl / minute for 35 minutes. Typically, 2000 RU was added. The biotinylated peptide (100 ng / ml) having the sequence indicated in the sequence listing as SEQ ID NO: 19 was passed over the streptavidin plate at a flow rate of 5 μl / minute for 2 seconds; 144 RU of the peptide was added to the streptavidin surface. The mAb 5109 at a concentration of 6.3 μg / ml alone or mixed with conventional concentrations of peptide 054 (SEQ ID NO: 19), or mixtures of 5109 and dilutions of samples with unknown amounts of collagen fragments, were passed over the Peptide surface for 1 minute at a flow rate of 10 μg / min. The slopes of the linear portions of the association phase for each curve were analyzed with the BIAevaluation ™ version 2.1 program. A standard curve of the competitive 054 peptide (SEQ ID NO: 19) was constructed against the slope. The amount of collagen epitope in the samples was determined by comparing the slope of a sample with the slopes of the standard curve to calculate the amount of epitope. Between each injection, the plate was cleaned with 0.1 N HCl for 30 seconds to remove the antibody.

TABLE 7 5109 Mixed with standard amounts of peptide 054 (SEQ ID NO: 19) Slope of the linear regression = -26.36 Intersection in y = -176 Sample: The supernatant of a digested by collagenase-3 MMP-13 of bovine nasal cartilage. The sample was tested on a BIAcore ™ plate diluted 2, 4 and 8 times. The calculated amounts of the collagen were determined in terms of the peptide 0.54 (SEQ ID NO: 19). The results appear in the fourth column of table 8. After multiplying by titration, the molar concentration (M) of the collagen can be determined in terms of the peptide pattern 054 (SEQ ID NO: 19).

TABLE 8 The titration and concentrations of the unknown sample in terms of the concentration of peptide 054 (SEQ ID NO: 19) The consistency of the results (last column) after correction for the dilution is shown by the agreement of the calculated values of three deferent dilutions Q'unto the last column) of the unknown samples. An average value of 75 mM type II collagen fragments is obtained for the supernatant of the bovine nasal cartilage.

EXAMPLE 6 Preparation of antibodies produced by genetic engineering related to 9A4 The basis for generating antibodies by engineering and their subsequent evaluation as important or biologically active molecules is cloning (with coupling in the appropriate configuration) and characterization of the V and VH domains of the parent antibody. The applicants have determined the structural sequences of V and VH of the treated antibodies and the exclusivity of the particular V-VH combination that forms the active binding site to the antigens described in this invention. Before cloning the genes of the variable region of 9A4, it was necessary to determine the protein sequence of portions of the variable domains of the parental antibody 9A4 IgG1, so that when the variable domains are cloned, it can be determined whether the correct variable domains have been obtained and not others derived from the myeloma fusion partner or an inactive pseudogene of the B cell when the hybridoma is generated. The supernatant of the culture containing 9A4 IgG1 was generated by culturing the hybridoma 9A4 in agitation bottles. The supernatants were adjusted to pH 7.5 with dibasic sodium phosphate and the salt concentration was adjusted with 3 M sodium chloride to a final concentration of 150 mM. The filtered supernatant (0.2 μ) was passed through protein G with a bed volume of 15 ml (Pharmacia) at a flow rate of 20 ml / min. After washing the column further with a solution of 150 mM NaCl, the antibody was eluted with 100 mM glycine pH 3.1. The antibody isotype was established using anti-sera from the mouse immunoglobulin isotyping assay kit (Boehringer Mannheim, Indianapolis, IN), and was found to be a murine IGg1 class antibody with a kappa light chain. It has been observed that some isolated proteins are "blocked" at their amino terminal end. "Blocked" means that the amino acid residue at the amino terminus of the polypeptide chain has been chemically modified with its structure post-translationally, by a cell action or some spontaneous chemical change in such a way that the polypeptide chain is resistant to the degradation of Edman The Edman degradation method is a chemical procedure that has been commonly used for the last 40 years to determine the amino acid sequence of proteins. The use of the Edman degradation technique, usually automatic installed in a laboratory instrument known as a protein sequencer or sequencer, is a conventional procedure known to those skilled in the art of protein biochemistry. A common mode of blocking is the conversion of an amino terminal glutaminyl residue to a pyroglutamyl moiety. This is produced by the cyclization of the glutaminyl moiety to form a structure that is inaccessible to the Edman reaction, because a new amide bond is formed between the alpha-amino group of the former and the delta-carboxyl group. Under these circumstances, the ability to obtain sequence information of the amino terminus of the protein depends on the elimination of the pyroglutamyl moiety by a chemical or enzymatic method. An important method is to use an enzyme called pyroglutamate-aminopeptidase (EC 3.4.19.3) to remove the pyroglutamyl moiety. The blocked protein, which may be in solution or electrotransferred to a membrane material such as PVDF (poly (vinylidene difluoride)), is treated with a pyroglutamateaminopeptidase solution until a sufficient amount of the protein has been unblocked to allow determination with success of the amino acid sequence by the automatic Edman chemistry. Examples of methods to accomplish this are described in Fowler et al., "Removal of N-Terminal Blocking Groups from Proteins" in Current Protocols in Protein Science, pp. 11.7.1-11.7.17 (Eds. Coligan et al.), Jonh Wiley, New York (1995). In the present work, the light and past chains of the mAb 9A4 were separated by polyacrylamide-SDS gel electrophoresis using a reducing agent (beta-mercaptoethanol) in the sample buffer. After electrophoresis, the polypeptides in the gel were electrotransferred to a PVDF membrane and detected by means of Coomassie brilliant blue staining R-250. The bands containing heavy and light chains of 9A4 were then cut off from the blot and treated separately with pyroglutamate-aminopeptidase. In each case, it was demonstrated that it was possible, after this treatment, to obtain information of the amino terminal sequence by Edman degradation. Sequencing was carried out on a Perkin-Elmer Applied Biosystems 494 Procise ™ protein sequencer. When it is desired to obtain information on the internal (i.e., non-N-terminal) amino acid sequence of the protein, the transferred samples of the protein can be digested with trypsin, and the resulting digest can be fractionated by HPLC to produce individual peptides which can then be sequence. The details of the works were the following.

N-terminal Unblocking with Pyroglutamate-aminopeptidase (PGAP) The antibody (9A4) was separated into its heavy and light chain constituents by SDS-PAGE on a 4-20% Tris-Gly polyacrylamide gel (Novex, San Diego). They were then electrotransferred to ProBlott ™ (Perkin-Elmer Applied Biosystems, Foster City, CA) and the bands were visualized by Ponceau S staining (Sigma). Membranes containing the light chain of 9A4 were counted and incubated for 30 minutes in a buffer containing 0.1 M sodium phosphate, 10 mM Na2EDTA, 5 mM dithiothreitol, 5% glycerol, and 0.1% Triton X-100. . Pyroglutamate-aminopeptidase (20 mg) (Boehringer Mannheim, Indianapolis, IN) was added to the vial, the contents mixed gently, and the reaction was incubated overnight at 37 ° C. The membranes were removed and washed thoroughly in water to remove all salts, detergent and enzyme. The membranes were then placed in the Applied Biosystems 494 protein sequencer (Perkin-Elmer) for analysis of the N-terminal sequence according to the manufacturer's instructions. The heavy chain was treated in the same way.

TABLE 9 Results of the N-terminal sequence of 9A4 protein sequencing The remains in parentheses indicate provisional data. Note 1: this sequence corresponds to residues 9 to 28 of SEQ ID NO: 33. Note 2: this sequence corresponds to residues 13 to 32 of SEQ ID NO: 32. In addition to the data of the N sequence -terminal obtained previously, the internal sequencing of the light chain of the antibody 9A4 was also carried out, according to the method developed by Fernández et al., Anal. Biochem., 218: 112-117 (1994).

Four bands corresponding to the light chain of 9A4 were cut from the ProBlott ™ and incubated for 30 minutes in a buffer containing 10% acetonitrile and 0.1% Triton X-100 in 0.1 M Tris-HCl, pH 8.8. Sequencing-quality modified trypsin (0.2 mg) (Promega, Madison, Wl) was added and the bands were incubated overnight at 37 ° C. The resulting peptides were extracted from the membrane by washing (with 60% acetonitrile and 0.1% TFA in H20) and sonication. Peptides were separated by reverse phase HPLC on a Vydac C18 218TP column (1.0 x 250 cm) (Vydac, Hesperia, CA). The peaks were hand picked in a visual manner, and the selected peaks were analyzed by automatic Edman sequencing in a model 494 protein sequencer as before.

TABLE 10 Results of the N-terminal sequence of protein sequencing of 9A4 peptide fragments The remains in parentheses indicate provisional data. Note 1: this sequence corresponds to residues 52 to 67 of SEQ ID NO: 33. Note 2: this sequence corresponds to residues 68 to 83 of SEQ ID NO: 33. Note 3: This sequence corresponds to residues 110 to 114 of SEQ ID NO: 33. The amino acid sequence of the light chain (VL-C aPPa) of mature 9A4 is as follows. 1 QrVLTQSPVF MSASPGEKVT MTCS.ASSSVS YMYWY QKPG SSPRLLIHAT SNLASGVPVR 61 FSGGGSGTSY SLTISRMEAE DAATYYCQQW RSYTRTFGGG EII TK * RADA 121 APTVSIFPPS SEQ TSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN S TDQDSKDS TYSMSSTLTL TSPIVKSFNR NEC 181 TKDEYERHNS YTCEATHKTS The underlined amino acids have been sequenced by automated Edman sequencing (see also Table 9 above). The sequence obtained from the cloned DNA, when compared to the amino acid sequences of the fragments of the original antibody protein shown above, indicates that the cloned gene is the correct one for the VL of 9A4 (comparison of the sequence derived from the sequencing of the DNA from cloned VL (see below) and murine Ckappa (obtained from Kabat et al., "Sequences of Proteins of Immunological Interest", US Department of Health and Human Services, NIH, 5th edition, publication No. 91-3242 , 1991) with the sequences obtained by Edman degradation). The above amino acid 106 is normally a Lys residue in the germ line segment J1, but has been mutated to the remainder shown above for the V of 9A4. Amino acid 106 marks the end of the VL domain, while amino acid 107 (Arg) marks the beginning of the kappa domain.

Cloning and determination of Vj_ and VH sequences of mAb 9A4 The hybridoma 9A4 cell line was cultured in HT medium (Sigma) with 5% fetal calf serum (Hyclone). MRNA was extracted from a cell pellet containing 1 x 10 7 cells using the Pharmacia Quick Prep ™ mRNA extraction assay kit (Pharmacia) according to the manufacturer's protocol. Then cDNA was synthesized for both segments of the V and VH gene using the Boehringer Mannheim cDNA assay kit (Indianapolis, IN) according to the manufacturer's protocol. The oligo used in the V cDNA reaction (referred to as MLK) and which appears in the sequence listing as SEQ ID NO: 21, is specific for the kappa region of the murine light chain, and the oligo used in the cDNA reaction of VH (referred to as MHG) is specific for a segment in the CH2 domain of the gamma region of the murine heavy chain. The MHG sequence appears in the sequence listing as SEQ ID NO: 22. This oligo was designed to anneal to the 4 murine IgG isotypes, including IgG1, IgG2a, IgG2b and IgG3. There is a single mismatch of bases for IgG1 in nucleotide 5 of SEQ ID NO: 22, but it will be apparent to those skilled in the art that this does not prevent re-association and subsequent generation of cDNA. The oligos were synthesized in Oligos etc. (Wilsonville, OR), Genosys (The Woodlands, TX) or Perkin-Elmer Applied Biosystems (Foster City, CA). The amino terminal amino acid sequence data was used to generate a possible oligo to isolate the correct 9A4 VH gene by PCR as follows. Based on the 20 amino acid sequence presented above for the amino terminal region of VH, which corresponds to residues 13 to 23 of SEQ ID NO: 32, and assuming that the terminal amine amino acid of the mature form of the antibody heavy chain segregated is actually a Gln residue (residue 12 of SEQ ID NO: 32), the known antibody sequences were compared with the VH of 9A4 using the Kabat database in "Sequences of Proteins of Immunological Interest", volume II, 1991 The V domains of the antibody that pair with the first 20 amino acids of VH of mature 9A4 include: mAb 264 (Nottenburg et al., Y Immunoi, 139: 1718-1736 (1987)); MAbRFT2 (Heinrich et al., J. Immunol., 143: 3589-3597 (1989)); and MAb2H1 (Li et al., Mol.Immunol., 27: 303-311 (1990)). Since it is important to obtain the original DNA sequence that corresponds to the amino terminal end of the mature antibody, the 5'PCR oligo must be designed so that it is reassociated 5 '(upstream) of the amino terminus, that is, in the segment of signal peptide. The DNA sequences of the above 3 antibodies, which are identical to the first 20 VH amino acids of 9A4, have known signal peptide DNA and amino acid sequences. The DNA sequences are shown here (underlined nucleotides indicate differences in sequence): 264: 5'- GG CTG TGG MC TTG CTA TTC (SEQ ID NO: 23) RFT2: 5'- GG GTG TGG ACC TTG CCA TTC (SEQ ID NO: 24) '2H1: 5'- GG GTG TGG ACC TTG CTA TTC (SEQ ID NO: 25).

These sequences encode amino acids -11 to -17 of the signal peptides for the VHs of the corresponding antibodies (see Kabat et al., Supra). The chosen 5'leaf, called MHMISC, was then designed to isolate the genuine 9A4 VH. The MHMISC sequence appears in the sequence listing as SEQ ID NO: 26. To isolate the V from 9A4, a set of five degenerate oligos was designed to anneal with the known murine kappa light chain conductive peptide segments. These oligos were used in 5 different PCR amplifications (see below). The sequences of the five degenerate oligos were obtained from The Dow Chemical Company (Midland, Ml) and are therefore known. The 5'oligo that produced a reliable 9A4 V was MK3, whose sequence appears in the sequence listing as SEQ IN NO: 27. These oligos condition residues -8 to -14 of the signal peptide.

Inverse primers were designed for VL and VH PCR from known sequences of the murine IgGI heavy chain constant region (from the CH1 domain) designated MIGG1 CH1, whose sequence appears in the sequence listing as SEQ ID NO: 28, and the constant region of the kappa light chain, called MLKN, whose sequence appears in the sequence listing as SEQ ID NO: 29. These oligos were placed (upstream) in relation to the 3 'primers used in the cDNA synthesis. The reassociation segment begins at nucleotide 10 of SEQ ID N0.29. The VH and V genes of 9A4 were amplified by PCR using the corresponding oligos described above, and 1 ng of hybridoma 9A4 cDNA. The PCR was performed using a GeneAmpR assay kit with native Taq-polymerase (Perkin Elmer) according to the manufacturer's instructions. The denaturation, annealing and polymerization temperatures were 94 ° C, 55 ° C and 72 ° C for 45, 45 and 60 seconds, respectively. For VH PCR and VCR, the annealing temperature changed to 60 ° C. PCR was carried out for 36 cycles plus a final polymerization cycle of 72 ° C for 7 minutes, followed by maintenance at 4 ° C. The PCR products were sequenced to determine and verify the DNA and amino acid sequences derived from the VL and VH. The oligos used for the determination of the sequence were MK3 and MLKN for VL, and MIGG1CH1 for VH, and appear in the sequence listing as SEQ ID NO: 27, 29 and 28, respectively. The DNA sequences corresponding to VH and V of 9A4 appear in the sequence listing as SEQ ID NO: 5 and 6, respectively. The amino acid sequences derived from the VH genes and VL of 9A4 appear separately in the sequence listing as SEQ ID NO: 32 and 33, respectively. Surprisingly, the oligo MHMISC (SEQ ID NO: 26) successfully delivered a VH sequence that exactly corresponded to the first 20 amino acids of the mature VH protein, determined by the sequencing of lgG1 9A4 proteins. Immediately 3 'of the 5'PCR oligo, MHMISC (SEQ ID NO: 26), the DNA sequence in the VH-conducting peptide segment of 9A4, is almost identical to the corresponding segment of mAb 2H1, and thus is likely to be derived from the same VH gene of the germ line as 2H1. There are only three differences in the amino acid sequence between the two antibodies in the VH regions; 1 on CDR1, 1 on CDR2 and 1 on FR3. These are probably somatic mutations that have occurred during the maturation process of affinity in these two antibodies, and may play a role in defining the specificity and affinity of each of the antibodies for their respective targets. The VH of 9A4 uses the murine JH2 binding segment gene and encodes residues 114 (within CDR3) to 126 of SEQ ID NO: 32.

The variable heavy chain of 9A4 belongs to family II of mouse Ig heavy chains from Kabat (the VH genes identified here were obtained from the Kabat database at http://immuno.bme.nwu.edu/famgroup. html). The VH heavy chain of 9A4 (SEQ ID NO: 5) closely resembles the ID No. 001246 of the data bank. There are only differences in these 2 VH genes, one that occurs in FR1 (silent mutation) and the other in CDR1 at amino acid position 45 (SEQ ID NO: 32), in which 9A4 has a lie and 001246 has a Met. It is very likely that both genes are also derived from the same germline VH. The methods of the present invention can also be carried out using VH genes in other antibodies related to the VH gene of 9A4 of the germline, so that when the VH gene product forms the VL-V pair of the productive antibody with the gene V of this other antibody, binds in an analogous way (in specificity and affinity) to 9A4. The VL gene of 9A4 and the derived amino acid sequences appear in the sequence listing as SEQ ID NO: 6 and 33, respectively. The amino acid sequence derived from the V-gene of 9A4 pairs with all the peptide fragments of the genuine 9A4 light chain protein sequence, as presented above. The variable light chain of 9A4 belongs to the XI kappa mouse family of Kabat, and is very similar to the ID No. 006306 of the database. There are 10 nucleotide mismatches, resulting in 7 amino acid differences. Two of these differences occur in FR1, 2 in CDR2, 1 in FR3, and 2 in CDR3. Since most of the changes occur in the CDRs, it is likely that these 2 genes are related because they are derived from the same V, or at least one very similar, in the germ line. The methods of the present invention can also be carried out using the VL genes of other antibodies derived or related to the V gene of 9A4 from the germline, so that when the VL gene product forms a VL-VH pair of productive antibody with the VH of this other antibody, it binds in an analogous way (in specificity and affinity) to 9A4. The methods of the present invention can also be carried out using antibodies in which VL and VH are derived from the VL and VH genes of the germline 9A4, described herein, so that when the products of the VL and VH form a productive VL-VH pair, this antibody or other than 9A4 binds essentially in an analogous manner (in specificity and affinity) to 9A4. The bases are now established from which to design and construct versions produced by genetic engineering of the antibody 9A4 of the invention. Below are two examples, both single chain antibodies (scFv). In one case, the design is VH-V-connector-HIS-MYC frames in the pCANTABd vector, and the other the scFv is constructed in the opposite orientation with a different connector, ie V -connector-VH-FLAG mark in the vector pATDFLAG. These examples are not intended to be limiting, but it will be apparent to those skilled in the art that the newly characterized 9A4 VH and VL domains (SEQ ID NO: 5 and 6) may be used in part or complete to obtain the types of antibodies previously described, such as Fab, or new configurations that use only a critical portion of the V. domains Construction of 9A4 scFv (Vjj-Connector-VL in PCANTAB6) SOE PCR primers (overlap extension cleavage) were used to prepare the assembly of the VH and VL fragments in an scFv antibody Two primers were designed and obtained in Perkin Elmer for the VH region: for the 5VH end primer an Sfi I site was added, while the 3 'end of the VH primer was added an overlapping sequence with the (Gly Ser) 3 linker. The 5VH primer was designated 9A4VH5CAN, and the 3'VH primer was designated 9A4H3CAN, which correspond to SEQ ID NO: 34 and 385 in the sequence listing For the V, a 5 'end primer was designed with an overlap in the Gly Ser linker region. , and a 3 'primer that incorporates a Not I restriction site, and were also obtained in Perkin Elmer The 5' primer was designated 9A4VL5CAN, and the 3V primer was designated 9A4VL3CANILE, which correspond to SEQ ID NO: 36 and 37 in the sequence listing.

The components of the VH and VL DNA were amplified by PCR and subsequently joined by an assembly SOE-PCR step. After the SOE-PCR reaction, a band was cut on an agarose gel, which was determined to be approximately 700 bp, and purified on the gel using the QIAquick ™ gel purification assay kit (QIAgen) according to manufacturer's instructions. The resulting DNA was digested with Sfi I and Not I, and used to couple to the pCANTABd DNA vector (obtained at Cambridge Antibody Technology, Melbourne, Cambridgeshire, UK), which was also digested with the same restriction enzymes. The coupled DNA was then used to transform competent E. coli TG1 cells by electroporation. The basic protocol for generating the competent E. coli TG1 cells is as follows. 500 ml of 2YT medium (Bio101, La Jolla, CA) preheated to 37 ° C in a 2 liter conical flask was inoculated with 2.5 ml fresh culture of TG1 cells grown overnight. The cells were cultured with vigorous aeration (200 rpm) at 37 ° C until the OD at 600 nm from 0.2 to 0.25, typically 1-1.5 hours later. The flasks were chilled for 30 minutes on ice, then the contents were poured into 250 ml centrifuge bottles and centrifuged at 4,000 rpm for 15 minutes in a pre-cooled Sorvall centrifuge (4 ° C) to pellet the cells. The cells were resuspended in the original volume of pre-cooled water, and re-centrifuged as before to pellet the cells.

The cells were resuspended in half the original volume, ie 250 ml, of pre-cooled water, and left on ice for 3 minutes, then resuspended and recentrifuged as before. The cells were then resuspended in 20 ml of pre-cooled 10% glycerol, transferred to a pre-cooled 50 ml Falcon tube, left on ice for 15 minutes, and centrifuged at 3,500 rpm at 4 ° C for 10 minutes in a centrifuge. of a table The cells were finally resuspended in 1.0-2.5 ml of precooled 10% glycerol and used directly in the electroporation stage. Coupled DNA (25-250 ng of vector pCANTAB6-SfiL / Not I with 9A4 VH-C-VL-SIII / Not I) was electroporated into the TG1 cells of E. coli components as follows. The coupled DNA was precipitated in ethanol by conventional protocols. The DNA pellet was dissolved in 10 μl of sterile deionized distilled water. The DNA (up to 10% of the total cell volume) was added to 100 μl of cells, and transferred to a pre-cooled electroporation cuvette (Biorad) and left on ice. The parameters of the Gene Pulser ™ electroporation apparatus (Biorad) were set to 25 μFD, 2.5 kV and the pulse controller was adjusted to 200 ohms The cuvette was dried with a paper tissue, placed in the electroporation chamber and pulsed once. Typically, time constants in the range of 3.5-4.8 msec were obtained. The electroporated cells were immediately diluted with 1 ml of 2YT medium supplemented with 2% glucose. The cells were transferred to a 15 ml Falcon tube and shaken at 37 ° C for 1 hour. The mixture of transformed cells (20-1000 μl) was placed on agar medium plates of appropriate size containing 2YT, 2% glucose and 100 μg / ml ampicillin (2YTAG). Initially, a clone was obtained (p9A4ICAT3-2) that had a 5 base insertion in the Not I site, which was taken from the frame to the downstream sequence. In order to correct it, a new oligo named 9A4NOTFIX3 was produced, whose sequence appears in the sequence listing as SEQ ID NO: 38. PCR amplification was carried out using E. coli cells containing p9A4ICAT3-2 as target for the annealing oligos, using the Advantage KlenTaq ™ Polymerase mixture (Clontech, Palo Alto, CA) according to the protocol manufacturer. The annealing oligos (35 pmol each) were 9A4NOTFIX3 and pUC19R. The sequence of pUC19R appears in the sequence listing as SEQ ID NO: 39; annealed upstream of the Sfi I site. The PCR cycles (in a Perkin Elmer Cetus model 9600 thermal cycler) were performed as follows: for the first cycle, the DNA denaturation was carried out for 1 minute at 94 ° C, the annealing for 45 seconds at 60 ° C, and polymerization for 1.5 minutes at 68 ° C. For cycles 2-31, the same times and temperatures were used, except that the denaturation times were reduced to 30 seconds. For the final cycle (32), the polymerization was carried out for 5 minutes, after which the cycler cooled the sample to 4 ° C. The TA cloning system (Invitrogen) was used to clone the resulting PCR products in plasmid pCR2.1 using the protocol suggested by the manufacturer. Twenty white colonies were investigated for inserts using the M13 Forward and M13 Reverse oligos (Invitrogen) (appear in the sequence listing as SEQ ID NO: 53 and 54, respectively) by PCR, using the KlenTaq ™ Polymerase as before. Three colonies produced inserts of the correct size in an agarose gel electrophoresis. One of them was chosen with the DNA sequence of the correct size for construct 9A4 and Vp-connector-VL, to work with it. The DNA insert containing the scFv gene was obtained by digestion of the pCR2.1 derivative with Sfi I and Not I, purified using the QIAquick ™ gel extraction assay kit and ligated with the vector pCANTAB6 digested with the same restriction enzymes. The DNA was introduced by electroporation into TG1 cells of competent E. coli as described above, and a clone named p9A4ICAT7-1 (ATCCD 98593) containing an active scFv was produced. The DNA sequence of this 9A4 scFv appears in the sequence listing as SEQ ID NO: 7, while the amino acid sequence appears separately as SEQ ID NO: 40. Note that the GTG codon, which starts at position 29 of SEQ ID NO: 7 is the initiation codon (Met). The sequence oligos used were pUC19R and FDTETSEQ, which appear in the sequence listing as SEQ ID NO: 39 and 41, respectively. See the information for SEQ ID NO: 7 and 40 in the sequence listing to obtain the specific characteristics of the DNA and the amino acid sequences for this scFv antibody. The scFv antibody of format 9A4 VH-linker-VL was expressed in TG1 cells of E. coli, and the antibody was purified using NTA-Ni agarose affinity chromatography (QIAgen), followed by Superdex 75 gel filtration chromatography to isolate the monomeric scFv species. The binding to the parental antigen of the scFv was evaluated in the BIAcore ™ system (see below). E. coli containing the plasmid pA4ICAT7-1 has been deposited in the American Type Culture Collection as ATCC 98593.

Construction of 9A4 scFv (VL-Connector-VFH) in pATDFLAG Oligos SOE were also prepared for the PCR-SOE assembly of the VH and VL fragments in the opposite configuration, ie V -connector-VH in relation to the example presented above which implies p9A74ICAT7-1. Two primers were designed for the V-connector region: at the 5 'end of the Neo I site and at the 3' end, an overlapping sequence was added to the 25 amino acid linker sequence that appears in the sequence listing as SEQ ID NO. : 42 (Pantoliano et al., Biochemistry, 30: 10117-10125 (1991)). The 5VL primer was designated 9A4VL5ATD and the 3'VL primer was designated 9A4VL3ATDILE. The sequences of these oligos appear in the sequence listing as SEQ ID NO: 43 and 44, respectively. For VH, a 5 'end primer was designed with overlap in the linker region and a 3' end PCR primer with an added Nhe I site. The 5'VH primer was designated 9A4VH5ATD, and the 3'VH primer was designated 9A4VH3ATD. The sequences of these oligos appear in the sequence listing as SEQ ID NO: 45 and 46, respectively. - The VL and VH components were amplified by PCR. The VL and VH were then joined in the linker region by SOE-PCR using oligos 9A4VLTATD (SEQ ID NO: 44) and 9A4VH3ATD (SEQ ID NO: 46). The correct size DNA, approximately 700 bp, was cut from a 1% agarose gel and eluted using a QIAquick ™ gel assay kit. The ends of the resulting DNA were cut with the restriction enzymes Neo I at the 5 'end and Nhe I at the 3' end. This was ligated with the expression vector pATDFLAG (PCT WO 93/12231) treated with the same restriction enzymes. Competent E. coli DH5a cells were transformed with the coupling according to the manufacturer's protocol, and placed on agar plates containing 20 μg / ml of chloramphenicol as a selection agent. The two clones that were sequenced, p9A4IF-5 and p9A4IF-69, did not have PCR or construction errors. The sequencing oligos were: UNIVLSEQ-5 '(SEQ ID NO: 30) and TERMSEQ (-) (SEQ ID NO: 31).

E. coli containing p9A74IF-5 was chosen for further investigation and the expression of engineered 9A4 scFv antibody. The DNA sequence of this scFv appears in the sequence listing as SEQ ID NO: 8, while the derived amino acid sequence appears separately as SEQ ID NO: 47. The specific characteristics of the V -C-VH-FLAG 9A4 scFv1, such as the signal peptide, the linker and the label locations are indicated in the sequence listing for SEQ ID NO: 8 and 47. Those skilled in the art are it will be apparent that the engineered antibody described could be expressed not only from E. coli, but also from other organisms, including but not limited to P. pastoris, baculovirus, Bacillus species, mammalian cells and the like. For the expression and purification of this scFv product from E. coli, cultures of 1-2 liters in LB broth containing 20 μg / ml chloramphenicol were cultured overnight at 37 ° C. The cells were pelleted in a Sorvall centrifuge using a GS-3 rotor. In the preparation of an affinity chromatography using an M2 affinity column (Kodak, New Haven, CT) (which is specific for the FLAG epitope shown in the sequence above), the sedimented E. coli cells were processed in a medium Tris / E DTA / rose out to isolate the periplasmic fraction, or sonicated (Soniprep sonicator) directly in a minimum volume of DPBS buffer. The affinity column was washed thoroughly with DPBS to remove any unbound material after loading the crude scFRv sample. The scFv antibody eluted using glycine 0.1 M-HCl, pH 3.1. The monomeric scFv species was isolated by Superdex-75 gel filtration chromatography (Pharmacia). The antibody was determined to be homogeneous by SDS-PAGE and Coomassie brilliant blue staining R-250. The antibody was quantified spectrophotometrically at OD 280 nm, it being defined that an absorbance of 1.4 is equivalent to 1.0 mg / ml of scFv, using a quartz cuvette with a step length of 1.0 cm. E. coli containing the plasmid p9A4IF-5 was deposited in the American Type Culture Collection as ATCC-98592. The antigen binding in the BIAcore ™ system was evaluated for the purified gene products of scFv p9A5ICATA7-1 and p9A41 F-5. A streptavidin plate was loaded with the biotinylated peptide of SEQ ID NO: 14 as in Example 1 above. It was shown that both scFv constructs bound to the antigen, ie, compared to the parental IgG 9A4, which binds with a K of 1.2 x 107 M, the 9A41 F-5 scFv had a K of 1.1 x 107 M. For 9A41CAT7-1 scFv, the output speed was 1.2 x 10"3 / seconds, compared to 1 J6 x 10" 2 / seconds for 9A4 IgG (parental antibody). These data indicate that the affinity of the engineered antibodies was at least as good or better than the parentals. As evidenced by the specificity and kinetic data determined by the BIAcore ™ system, the two engineered antibodies containing the VL and VH domains of 9A4 described above have the desired characteristics for their use in the measurement assays. quantitative described in examples 3 and 4 above.

Therefore, other engineered antibodies formed by some or all of the VL and VH of 9A4 described herein, which have the affinity and specificity of the parental 9A4 antibody, will be considered useful in the methods of the present invention.

EXAMPLE 7 Preparation of the antibodies produced by genetic engineering related to 5109 Before cloning the genes of the variable region of 5109, it is necessary to determine the protein sequence of portions of the variable domains of the parent 5109 antibody, so that when the domains are cloned, it can be determined that the correct variable domains have been obtained and not other derivatives of the companion of fusion of myeloma or an inactive pseudogene of the B cell used when the hybridoma is generated. The supernatant of the culture containing 5109 was generated by culturing the 5109 hybridoma in agitation bottles. The supernatants were adjusted to pH 7.5 with dibasic sodium phosphate and the salt concentration was adjusted with 3 M sodium chloride to a final concentration of 150 mM. The filtered supernatant (0.2 μ) was passed through G protein with a bed volume of 15 ml (Pharmacia) at a flow rate of 20 ml / min. After washing the column further with a solution of 150 mM NaCl, the antibody was eluted with 100 mM glycine, pH 3.1. The isotype of the antibody was established using antisera from the mouse immunoglobulin isotyping assay kit (Boehringer Mannheim), and was found to be a murine antibody to cale lgG1 with a constant kappa light chain domain. In the present invention, light and heavy chains of mAb 5109 were separated by SDS-PAGE using a reducing agent (beta-mercaptoethanol) in the sample buffer. After electrophoresis, the polypeptides in the gel were electrotransferred to a PVDF membrane and detected by Coomassie brilliant blue staining R-250. The bands containing the 5109 heavy and light chains were then cut and subjected to Edman degradation. The sequencing was carried out in Perkin-Elmer Applied Biosystems model 494 Procise ™ protein sequencer, according to the manufacturer's protocols. The sequences of the heavy and light chains that were obtained appear in table 11, and correspond to residues 1 to 40 of SEQ ID NO: 48 for VH, and residues 1 to 39 of SEQ ID NO: 49 for the VL.

TABLE 11 Results of the N-terminal amino acid sequence of 5109 Provisional amino acids are indicated by a parenthesis. Note: position 22 on VH and position 23 on V are normally Cys, and can not be determined by Edman degradation.

Cloning and determination of the VH and V_ sequences of mAb 5109 Hybridoma cell line 5109 was cultured in HT medium (Sigma) with 5% fetal calf serum (Hyclone). The cells were pelleted (2.5 x 10 7 cells / pellet) and frozen at -80 ° C until use. The extraction of the mRNA (Oligotex ™ direct mRNA assay kit, QIAGEN) is carried out according to the manufacturer's instructions. The cDNA was then synthesized for the VL and VH regions using the synthesis assay kit of the first cDNA strand of Boehringer Mannheim. The oligo using in the VL cDNA ratio was specific for the kappa region of the murine light chain (MLK), while the oligo used in the VH cDNA reaction was specific for a segment in the gamma CH2 region of the chain heavy murine. The sequences of oligos MLK and MGH appear in the sequence listing as SEQ ID NO: 21 and 22, respectively. The PCR primers were designed for the N-terminal sequence of the mature and segregated forms of the heavy and light chains, based on the amino acid sequences that were obtained for VH and V by Edman degradation. The sequences were compared with the Kabat database; it was found that VH of 5109 was similar to the members of Kabat's IIID subgroup, while V of 5109 was very similar to members of Kabat's subgroup II.

The sequences of the 5'VH primer and the 5'VL primer were designated 51-09VH'NDe and 51-09V 5'NDe, respectively, and appear in the sequence listing as SEQ ID NO: 50 and 51. Inverse primers were designed. from known sequences of the heavy chain constant region of lgG1 (for a segment in the CH1 domain) and the constant region of the kappa light chain. Both 3'oils are found 5 '(upstream) of the original oligos used to generate the cDNA. The 3'V primer (designated MIGG1CH1) and the 3'V primer (designated MULK2) appear in the sequence listing as SEQ ID NO: 28 and 52, respectively. The resulting PCR products for VH and V were coupled in pCR2.1 and the representative clones were chosen for subsequent DNA sequencing. DNA sequencing was carried out in an Applied Biosystems model 373 stretch sequencer, which was adjusted and managed according to the manufacturer's protocols. For the determination of the DNA sequence, the commercial sequence oligos of Invitrogen M13F and M13R were used, whose sequences appear in the sequence listing as SEQ ID NO: 53 and 54, respectively. It was found that the first 21 amino acids that were determined by Edman degradation for the mature amino terminal ends of VL and VH of 5109 were identical to the corresponding amino acid sequences of the DNA sequence of the corresponding genes that were cloned by PCR.

The DNA sequences of the VH and V domains of 5109 appear in the sequence listing as SEQ ID NO: 10 and 11, respectively. The VH and V amino acid sequences of 5109 appear separately in the sequence listing as SEQ ID NO: 48 and 49, respectively. It should be noted that although the DNA, as presented, encodes the correct amino acids in the amino terminal segments of each of the VH and VL segments that correspond to the PCR reassociation oligos, the exact codons for the amino acid segments of the antibody that correspond to these oligos, as they would appear in the DNA of the original hybridoma, are not unambiguous. The VH of 5109 uses the JH3 binding segment gene with 2 mutations in the codon, which would otherwise encode a Thr moiety (positions 328 and 330 of SEQ ID NO: 10), but that in the VH of 5109 is a Wing rest (position 110 SEQ ID NO: 48). The variable heavy chain 5109 is closely related to the XIV heavy chain family of Kabat Ig, and closely resembles the ID No. 002754 of the database. There are 24 differences in nucleotides between these two VH genes, resulting in 14 amino acid differences. It is very likely, based on this large number of differences, that these two genes are not derived from the same germline gene. However, it may be possible that the two are derived from the same germ line and that both have mutated in the maturation process of the affinity in vitro, and that the resulting divergence has been amplified.

The methods of the present invention can also be carried out using the VH genes in other antibodies related to the VH gene of 5109 of the germline, so that when the VH gene product forms a VL-V pair of antibody that is productive with the V of this other antibody, binds in an analogous manner (in specificity and affinity) to 5109. The VL of 5109 used a J5 binding segment and codes for amino acids 102 to 112 of SEQ ID NO: 49. The CDR3 of this antibody it is relatively rare, because of the rest of Cys-94 it contains. The CDR3 extends from the remainder 94 to 102 of SEQ ID NO: 49. When the 5109 single chain antibodies were engineered with a VL of 5109 (see below), two versions were made, one with the Cys- 94 parental and others with Be replacing Cys-94. Ei V of 5109 belongs to the VI kappa family of Kabat mouse, and is very similar to n ° ID 005481, 005842, 005843 and 005844 of the database. The four antibodies in the database are identical in their nucleotide sequences, so a comparison with VL of 5109 can be established with all four at the same time. There are 12 nucleotide mismatches, which result in 8 amino acid differences. One of these differences occurs in FR1, 3 in CDR1, 1 in FR2, 1 in CDR2, 1 in FR3 and in CDR3. These changes occur throughout the VL, but accumulate in the CDRs, with 5 of the 8 differences in these hypervariable segments. Therefore, it is very likely that these genes are related because they derive from the same VL, or from a very similar one, from the germ line. The methods of the present invention can also be carried out using the VL genes in other antibodies related to the V gene of 5109 of the germline, so that when the product of the V gene forms a VL-VH pair of antibody that is productive with the VH of this other antibody, binds in an analogous manner (in specificity and affinity) to 5109. The methods of the present invention can also be carried out using antibodies in which VL and VH of 5109 of the germline, described in FIG. present, so that when the products of the VL and VH genes form a productive VL-VH pair, this antibody or a different one of 5109 binds essentially in an analogous manner (in specificity and affinity) to 5109.

Construction of engineered antibody 5109 scFv fyH-linker-Vi) in pUC119 SOE PCR primers were used to prepare the VH and V components for assembly. All oligos used in the construction of 5109 were synthesized by the inventors, in a Beckman Oligo 1000M DNA synthesizer (Fullerton, CA). These five PCR primers, designated 5109 VH 5 ', 5109 VH 3', 5109 VL 5 ', 5109 VL 3' SER and 510 VL 3'CYS appear in the sequence listing as SEQ ID NO: 55, 56, 57, 58 and 59 respectively.

Oligo 5109 VL 3'Ser (SEQ ID NO: 58) was used to change the Cys-94 from SEQ ID NO: 49 to Ser-94 in the scFv construct (corresponds to the Cys residue at position 248 of SEQ ID. NO: 63) to potentially improve the stability of the resulting scFv. Another primer, 5109 VL 3'Cys (SEQ ID NO: 59) was also synthesized in order to maintain the original Cys sequence. After the SOE reaction, the DNA was amplified by PCR and the products were directed with Sfi I and Not I for subsequent coupling with the expression vector pUC119, or they were directly coupled to the sequencing vector pCR2.1. The coupling products of pUC119 were transformed into competent DH5a cells, whereas the pCR2.1 couplings were transformed into InfVa 'competent cells. Individual clones were investigated by PCR using the oligos pUC19R and mycseqIO. The DNA of the positive clones was subjected to sequencing. The DNA sequence was verified using an Applied Biosystems model 373 stretch sequencing unit, according to the manufacturer's instructions. The following oligos were used for the sequencing of engineered antibodies 5109 scFv potential, directly coupled in pUC119: pUC19R, MycSeqIO, Gly4Ser5 ', Gly4Ser3 \ whose sequences appear in the sequence listing as SEQ ID NO: 39, 60, 61 and 62, respectively. For the 5109 DNA modules assembled in the pCR2.1 vector, oligos M13F and M13R obtained in invitrogen (SEQ ID NO: 52: and 53) were used for the sequence priming reactions, together with SEQ ID NO: 61 and 62, as two internal oligos. The clones directly harvested to pUC119 contained a large number of PCR errors. However, an acceptable clone (H55) was identified in the pCR2.1 vector. This construct was then subcloned by digestion of scFv with Sfi I and Not I, and coupling in a similarly digested pUC119 vector. The clones were investigated to detect the insert by PCR amplification using the oligos pUC19R and MycSeqIO (SEQ ID NO: 39 and 60). Three clones were subjected to sequencing. A clone with the desired sequence was identified and named p5109CscFv7 (ATCC 98594); the DNA and the derived amino acid sequences appear in the sequence listing as SEQ ID NO: 9 and 63, respectively. The sequence characteristics of this engineered antibody appear in SEQ ID NO: 63. When the 5109 scFv was being generated by PCR, a mutation (a PCR error) occurred in nucleotide 738 of SEQ ID No: 9, changing the amino acid encoded by the altered codon from valine to alanine. This corresponds to the Ala residue at position 237 of SEQ ID NO: 63 (the sequence of 5109 scFv). As it is a conservative difference, it was not considered important in terms of affecting the activity of the products created by genetic engineering, and therefore it was allowed to remain in the VL of the described scFv constructs. Of course, it will be apparent to those skilled in the art that this PCR error can also be corrected and obtain the original Val residue in that position.

To verify that the new single chain constructs maintained the binding properties of the parent molecule, 5109 scFv was expressed in E. coli and purified. An initial 2YT culture (50 ml) containing 100 μg / ml ampicillin and 2% glucose was inoculated with 50 μl of glycerol stock at -80 ° C. the cultures were incubated overnight at 30 ° C with shaking at 300 rpm. Each of the six 2 liter flasks containing 2YT medium supplemented with 100 μg / ml ampicillin and 2% glucose was inoculated with 5 ml of the initial culture incubated overnight. The cultures were incubated at 30 ° C until they became turbid, and were subsequently induced by adding IPTG (B-D-isopropyl thiogalactopyranoside, Boehringer Mannheim) to a final concentration of 1 mM. The cultures were cultured for a further 4 hours for the production of scFv. The cells were centrifuged at 5000 x g for 10 minutes. Cell pellets were stored at -20 ° C until processed. For the purification of scFv, the cell pellets were resuspended in TES (0.2 M Tris-HCl, 0.5 mM EDTA, 0.5 M sucrose). after resuspension, a 1: 5 dilution of the previous TES buffer containing protease inhibitors (Complete Protease Inhlbitor Cocktail, Boehringer Mannheim) was added. This preparation was left in incubation at 4 ° C for 30 minutes. After incubation, the cells were pelleted at 12,000 x g for 15 minutes. MgCl2 was added to the resulting supernatant until a final concentration of 5 mM was reached. Ni-NTA agarose (QIAgen) 1 x was washed in PBS, pH 7.4, containing 300mM NaCl, 15mM imidazole and 0.2% Tripton-X. The washed agarose was then added and the suspension was left in incubation for 30 minutes at 4 ° C. Then Ni-NTA + scFv agarose spheres were washed four times as previously described. The scFv was eluted in washing buffer containing 250 mM imidazole. The eluate was desalted on a NAP-25 column (Pharmacia-Biotech, Uppsala, Sweden) and concentrated using a Centriprep ™ concentrator device (Amicon, Beverly, MA). The products were electrophoresed on SDS-PAGE and visualized by silver staining. The resulting scFv products for p5109CscFv7 (Cys) and p5109SscFvA9 (Ser) were approximately 15% and 75% pure, respectively. Using the Origin ™ methodology (technical manual, Origin ™ instrument, Igen Corporation, Gaithersburg, MD), the binding to the biotinylated peptide 225 (prepared by Anaspec, Inc., and having the sequence appearing in the sequence listing as SEQ ID NO: 64) by the species 5109 scFv. The biotinylated peptide 225 was added to 800 μg / ml streptavidin-coated magnetic spheres (Dynabeads ™, Igen, Gaithersburg, MD) to a final concentration of 10 mM, and incubated for 15 minutes. The new engineered scFv antibody was added to the peptide / spheresolution for 30 minutes with agitation. Anti-rutheniated myc brand 9E10 mAb was added, and the solution was incubated for 30 minutes, then 200 μl of the Igen assay buffer (Igen, Gaithersburg, MD) was added, and the ECL signal was read on the Origin ™ instrument ( Igen). The 9E10 mAb was generated and purified from the 9E10 cell line, which was obtained from the ATCC. The mAb was routinized using the N-hydroxysuccinamide Origin ™ TAG-NHS Ester derivative (Igen), according to the manufacturer's instructions. In the absence of 5109 scFv, a background effect signal of 2995 ECL units was obtained. The addition of construct 5109 scFv Cys (p5109CsFv7) yielded 536,997 ECL units. The addition of construct 5109 scFv Ser (p5109SscFvA9) resulted in 694,253 ECL units. These results demonstrate that both biologically active 5109 scFv antibodies were biologically active and bind to the same peptide fragment related to the collagen to which mAb 5109 binds. A culture of E. coli containing p5109CscFvA9 was deposited in the American Type Culture. Collection as ATCC-98594. The specificity data determined by Origin ™ technology demonstrate that the engineered antibody containing the VL and VH domains of 5109 described above, has the desired characteristics for use in the quantitative measurement tests described in examples 4 and 4 above. Therefore, it is considered that other engineered antibodies, formed by part or all of the VL and VH of 5109 described herein, are useful for practicing the methods of the present invention. It should also be noted that the engineered antibodies formed by a combination of the VH and VL domains of 9A4 and 5109, as a bispecific scFv dimer of composition: 5109VL-connector-5109VH-connector-9A4VL-connector-9A4VH-label (s) they would also be useful, as the only antibody reagent in examples 3 and 4 above. The difference would be that a single bispecific reagent could be used in an ELISA stage, instead of using 9A4 and 5109 separately. It will be apparent to those skilled in the art that a series of genetic compositions comprising the variable domains of the present antibodies can be linked to form a variety of bispecific molecules, and thus simplify the assays presented in Examples 3 and 4 The avidity of said molecules can be such that the overall sensitivity of the assay can also be significantly improved.

EXAMPLE 8 Mutations or differences in the amino acid sequences of the antibodies can maintain the binding properties (affinity and specificity), and thus the usefulness of the parent antibody. This is demonstrated below by generating a series of mutants in the CDR3 of VH 9A4. It will be apparent to those skilled in the art that other mutations in other regions of the VL and VH genes of the same germline of 9A4 and 5109, can produce antibodies with the same or better binding properties, relative to the original antibodies described. in this invention.

Generation of mutants of the VH CDR3 region of 9A4 The pCANTABd derivative of 9A4 scFv, designated p9A4ICAT7-1 presented in Example 6 above, was used as a starting material to generate mutants in the CDR3 segment and the Vernier residues immediately adjacent to the CDR3. The parental DNA sequence of the CDR3 VH region, which is the target for the mutation, comprises nucleotides 383 to 409 of SEQ ID NO: 7. The derived amino acids corresponding to these nucleotides are residues 119 to 127 of SEQ ID NO: 40. CDR3 begins at residue 121 and ends at 126. To introduce random mutations in this area, the VH and VL regions were amplified separately by two PCR. In the first PCR, the oligos (obtained in Olígos Etc.) pUC19R (SEQ ID NO: 39) and 9A4MUT (SEQ ID NO: 65) were used to amplify the VH portion, being 9A4MUT the oligo that introduces the mutations. Nucieotides 25 to 51 to 9A4MUT (SEQ ID NO: 65) were 10% seeded. In other words, the sequence as written represents 90% of the nucleotides added in each 25-51 position, while the other 3 nucleotides, in each position from 25 to 51, were introduced into the up-growing oligo chain at 3.3% each . This was carried out by methods well known to those skilled in the art of nucleotide synthesis. The fact that random nucleotides have actually been introduced in this defined region will be discussed below. The second PCR used the two obligos (also obtained in Oligos Etc.) 9A4L5 (SEQ ID NO: 66) and FDTETSEQ (SEQ ID NO: 41). This produced the VL-linker portion to the Not I site in p9A4ICAT7-1 at the 3 'end with VH overlap at the 5' end, which allows subsequent coupling and assembly with the mutated VH PCR products, as in first PCR. The PCR was carried out as follows. For the VH mutant, 50 pmol of each of the oligos pUC19R (SEQ ID NO: 39) and 9A4MUT (SEQ ID NO: 65) were used in 100 μl reactions. The template DNA target was an aliquot of DNA-p9A4ICAT7-1 purified by SNAP (Invitrogen). Taq-polymerase (Perkin Elmer) was used in the 30 cubits of the PCR. The times and temperatures of the denaturing, coupling and reaction of the polymerase were 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes, respectively. For the 30 ° C cycle, the polymerization reaction was extended for a total of 10 minutes, before cooling the reaction to 4 ° C. For the VL, which would overlap with the VH species, the PCRs were carried out using KlenTaq ™ (Clontech) and Taq-polymerase (Perkin Elmer) polymerase. The DNA products were purified in the QIAgen gel extraction assay kit. The PCR assembly reaction for the VL and the mutated VH was carried out using aliquots (1-2 μl) of each of the purified PCR products in 25 total cycles / 2 temperature: 94 ° C for 1 minute, 65 ° C for 4 minutes and maintenance at 4 ° C at the end. No oligos were used at this stage. For the PRC of the mutated 9A4 assemblies, 5 μl of the reaction of unpurified assemblages as a template was used, and the oligos pUC19R (SEQ ID NO: 39) and FDTETSEQ (SEQ ID NO: 41) as assembly primers. Products of correct size were observed at approximately 900 bp and gel purified using the QIAgen gel extraction assay kit. The mutated 9A4 scFv inserts were treated with Sfi I and Not I to prepare them for coupling with the pCANTABd DNA vector cut with the same restriction enzymes. After the coupling, the DNA mixture was precipitated in ethanol and dissolved in 24 μl of sterile deionized distilled water. Precipitation of competent E. coli TG1 cells was carried out as described in Example 6. Twelve electroporations were performed separately and finally were pooled and plated. A total of 1.5 x 106 clones were obtained and stored as a glycerol stock solution at -80 ° C. A random sampling of the 12 clones indicated that 9 of them (one clone did not produce sequence data) had an insert when they were investigated by PCR, using the oligos FDTETSEQ (SEQ ID NO: 41) and pUC19R (SEQ ID NO: 39) . These inserts were purified using the QIAgen assay kit and the VH CDR3 sequence was determined. The results of the sequencing data are presented below. The DNA sequence of the "parental sequence" appears in the sequence listing as nucleotides 383 to 409 of SEQ ID NO: 7. insert sequences page 98 Sequence 5'- GCT AGG GGC GGT AGC CTT GAC TAC TGG - 3 'parental: 9A4MUT-1: 5'- GCT CGG GGC GGT AGC CTT GAC TAC CGG - 3 '2 9A4MUT-3: 5'- GCT GGG CCC TGT ATC CTT GAT TAC TGG - 3' 6 9A4MUT-4: 5'- GCT ACG GGA GGT AGC CTT GAC TAC TGG - 3 '2 9A4MUT-6: 5'- GTT TGG GGC GGC AGC CCT GAC CAC AGG - 3"6 9A4MUT-7: 5'- GCT TGG GGC GGC AGG TAT GAC TAC TGG - 3 '5 9A4MUT-8: 5'- GCT ANG GTC AGT AGC CTT GAC TCC TGG - 3 '3 9A4MUT-10: 5 * - GCT ACG GGC TGT AGT CAT GAC TAC CGC - 3 '6 9A4MUT-12: 5'- GCT AGG GGT GGT AGC CTT GAC TAC TGG - 3 '1 Mutations in the previous group vary from 1 to 6 in each clone, and occur in different positions. Mutations are indicated underlined in the various previous clones (N = the nucleotide was not known). Only one clone (9A4MUT-12) of the 8, tested in this way, produced the parental amino acid sequence. Based on the randomness of the results shown above, a good mutated bank was generated. Therefore, a selection was made with biotin to find those that bind peptide 040 (SEQ ID NO: 14), which is the epitope of 9A4.

Description of Biotin Selection of the Antibodies Obtained Next An aliquot of approximately 100 μl of the mutated bank strains was added to 25 ml of 2YT medium containing 100 μg / ml ampicillin and 2% glucose. The culture was incubated at 37 ° C for approximately 60 minutes, or until the cells reached the semilogarithmic phase (ODß or nm = 0.5 to 1.0). Then the helper phage M13K07 (Pharmacia, Uppsala, Sweden) was added to the culture to a concentration of 5 x 108 pfu / ml. then the auxiliary phage was allowed to infect the culture for 20 minutes at 37 ° C without agitation, and then for an additional 25 minutes at 37 ° C with agitation at 200 rpm. The infected cells were transferred to a 50 ml conical centrifuge tube, and pelleted at 300 rpm for 10 minutes. The cells were resuspended in 2YT medium containing 100 μg / ml ampicillin and 50 μg / ml kanamycin. This culture was transferred to a fresh 250 ml flask, and incubated at 30 ° C for 2 hours, during which phage particles were produced. The cells were removed by centrifugation at 14,000 rpm for 2 minutes. Then aliquots (1 ml) of the phage were blocked for 30 minutes at room temperature by the addition of PBS and NFDM to a final concentration of 1 X PBS and 3% NFDM. This was accomplished by the addition of 200 μl of 6 x PBS, 18% NFDM solution, to 1 ml of the phage. 040 biotinylated peptide (SEQ ID NO: 14) was added to the phage solution at concentrations ranging from 10 pM to 1 μM. This solution was incubated for 60 minutes at room temperature. Streptavidin coated magnetic beads (Dynal, Oslo, Norway) were blocked at room temperature with shaking with inversion in 3% NFDM in PBS. After incubation, the streptavidin spheres were captured on the sides of the tube with a magnet, and the blocking solution was carefully removed by aspiration. The phage blocked with the bound peptide was then added to the streptavidin spheres, and allowed to incubate at room temperature with inversion shaking for 15 minutes. The complexes of the spheres were captured magnetically and the unbound phage was carefully removed by aspiration. The complexes of the spheres magnetically bound were washed 4 x with PBS containing 0.1% TW-20 and 4 x with PBS only. After the final capture, the complexes of the spheres were resuspended in 100 μl of triethylamine with 100 mM in PBS and neutralized with an equal volume of 1 M Tris, pH 7.4. The E. coli TG1 cells in semilogarithmic phase (10 ml) were then infected with 100 μl of complexes from the spheres. The infection was allowed to progress for 20 minutes at 37 ° C without agitation, and then for another 25 minutes at 37 ° C with shaking at 200 rpm. The infected cells were then pelletedwere resuspended in 500 μl of fresh 2YT medium and 500 μl were placed on 243 x 243 mm 2YT agar plates containing 100 μg / ml ampicillin and 2% glucose. The plates were incubated overnight at 30 ° C. After approximately 16 hours of growth, the colonies were recovered by scraping and used to inoculate the liquid cultures. The process was repeated for a minimum of two and a maximum of five rounds of selection. Clones recovered from biotin selection were cultured and scFv production was induced, which was purified according to the protocol summarized below.

Preparation of mutant clones of scFv using the hypotonic shock method The culture was pelleted and resuspended in 0.8 ml of ice-cold TES buffer (0.1 M Tris-HCl, 0.5 nM EDTA, 0.5 M sucrose). TES (1.2 ml of ice cold 1: 5 dilution) was added and the culture was incubated on ice for 30 minutes. The cells were pelleted at 4 ° C at 14,000 rpm (30 min). The supernatant of scFv was added to a fresh tube containing 10 μl of 1.0 M MgCl 2. NTA-agarose (200 μl) (QIAgen) was prepared by washing in washing buffer midazole phosphate containing 50 nM Na phosphate, pH 8.0 NaCI 500 mM, 20 mM midazole, and 0.1% TW-20. The supernatant of scFv was added to the NTA-agarose, and incubated at 4 ° C for approximately 30 minutes. The NTA-agarose with scFv was stirred and 500 volumes of elution buffer were added. The elution buffer consisted of 50 nM Na phosphate, pH 8.0, 500 mM NaCl and 250 mM imidazole. The eluate was vortexed and centrifuged to remove the NTA-agarose. The scFv supernatant was subjected to a buffer exchange by passing it over a NAP-5 column (Pharmacia), according to the manufacturer's instructions.

Measurement of the exit velocities of the scFv constructs The exit velocities of the scFv constructs were measured by analyzing the dissociation data obtained in the system BIAcore ™ (Pharmacia Biosensor) with the BIAevaluatíon ™ version program 2. 1. To obtain data from the BIAcore ™ system, streptavidin surfaces were prepared on BIAcore ™ plates, as previously described in Example 1. A biotinylated peptide (SEQ ID NO: 14) was attached to the streptavidin surfaces prepared up to RU densities that vary from approximately 2-11 RU / surface. The lower level of derivatization helps to avoid erroneous output speeds, which can occur if mass transport is a problem. The purified scFvs were injected onto these surfaces to allow binding of the constructs for 60 seconds. The PBS buffer was then replaced only and the scFv dissociation was allowed to proceed for a further 280 seconds. The exit velocities were calculated from this dissociation data.

TABLE 12 Summary of mutant VH sequences of CDR3 of 9A4. Amino acid sequences determined from the results of DNA sequencing. The parental sequence for clone ICAT7-1 corresponds to residues 118 to 127 of SEQ ID NO: 40.

Note: the above ICAT-7 and IF-5 clones show the CDR3 regions that have the parental sequence. It is evident from the data presented above that changes in the amino acid sequence of the parent antibody can be made, while binding to the target is maintained. Although the differences in the exit rates of the antibodies presented above are within an order of magnitude, by using different regions of VH or VL of the antibody for the mutations, or by discovering different antibodies obtained by immunization having VL domains and / or V H derived from the same genes of VL and VH of the germinal line of 9A4 or 5109, antibodies with variable or improved binding properties can be discovered in relation to the parental antibodies described in examples 6 and 7 above.

EXAMPLE 9 Description of a sandwich sandwich optimized for urine samples When testing biological fluids from a patient to detect the presence of TUNE fragments, it is preferred to use urine as a biological test medium. Urine is extremely easy to collect, and collection is done non-invasively. In addition, the epitope should be more concentrated in the urine than in the blood. And most importantly, urine is less likely to contain proteins that inhibit the assay than blood. Therefore, it is important to develop a test protocol that optimizes the measurement of TUNE fragments in the urine. In a preferred protocol in this, mAb 5109 is used as capture antibody, and mAb 9A4 as detection antibody. It is preferred to use each mAb in these papers, while 9A4 is able to weakly bind fragments derived from type I and III collagen, in addition to the desired type II collagen fragments. Since types I and III are present in the human body in considerable excess compared to type II collagen, fragments of types I and III are present in large excess in urine when compared to type II fragments (approximately 1, 000 times more, see Kivirikko, nt Rev. Connect, Tissue Res., 5: 93-163 (1970)). Therefore, although 9A4 binds more strongly to type II, it continues to significantly bind to types I and III in urine, largely due to the presence of excess amounts of these other collagen fragments. It has also been discovered that unknown components of the urine can interfere with the test results, producing erroneous readings. In an effort to reduce this effect, it has been determined that the control samples constructing the standard curve are preferably prepared from a serial diluted standard solution of concentrated TUNE fragments (preferably peptide 131, SEQ ID NO: 67) diluted in control urine gathered. The pooled control urine is a mixture of urine specimens from multiple subjects, in which each sample has been previously determined to contain extremely low levels of TUNE fragments, preferably very extremely low. By dilution of the control TUNE peptide in the pooled control urine, the effects caused by proteins or other components of the urine will be recreated in the control curve, and will produce a more accurate curve for the test samples. In another step to reduce false effects caused by urine, magnetic spheres are used immediately prior to analysis in the Origin ™ analyzer to remove materials that have non-specifically bound during the test. After the control and assay samples have been incubated with the antibodies (including the biotinylated capture antibody), the magnetic streptavidin spheres are added and a magnet is used to keep the spheres inside the test tube while removing the fluid. The spheres are then resuspended in a buffer solution before measuring the signal generated by the detection antibody. For optimal binding of the antibody to the TUNE fragments, it is preferred that the assay buffer maintain a pH of about 7.5. The protocols published for the use of the Origin ™ system employ a test buffer with 10 mM Tris, pH 7.5. After attempts to analyze urine specimens gave lower than optimal results, it was determined that 10 mM resulted in an insufficient concentration of Tris to maintain the pH at approximately 7.5. The tests determined that the optimum concentration of Tris is between about 50 and about 250 mM, while a concentration of about 100 mM is more preferred in the present. In the preferred embodiment herein, the assay buffer is TTBN-100, which consists of 100 mM Tris, pH 7.4, 1% Tween-20, 1% BSA and 140 mM NaCl. Any buffer that keeps the samples at an appropriate pH and does not interfere in any way with the results can be used. It is important to have the appropriate concentration of antibodies in order to achieve an adequate measurement of the amount of TUNE fragments present in the sample. A preferred concentration of 9A4 is from about 10 to about 30 μg / ml, with a concentration of about 20 μg / ml being preferred therein. A preferred concentration of 5109 is from about 5 to about 20 μg / ml, a concentration of about 10 μg / ml being preferred here. Finally, the standard curve for the TUNE test is linear in a defined interval. Typically, this range will be between about 1.3 ng / ml (i.e., about 20 fmol per reaction) at about 42.5 mg / ml (i.e., about 625 fmol per reaction). As such, control samples containing known amounts of TUNE will preferably be prepared at concentrations ranging from about 1.3 to about 42.5 ng / ml of TUNE fragments. The unknown test samples are preferably assayed in duplicate, at full concentration, and if necessary diluted in control urine at various levels, so that it is likely that at least one concentration of each test sample is within the linear of the control curve. In a preferred assay, the capture antibody is biotinylated and linked to a magnetic streptavidin sphere after incubation with the sample. The IGEN Origin ™ system is a preferred method here for measuring the detection antibody signal. When this methodology was used for the first time, the washed magnetic beads were resuspended in the Origin ™ assay buffer. Surprisingly, it has been found that resuspension of the beads in this buffer can lead to instability of the sandwich complex of the antibody, and displace the test results. This problem can be solved by quickly testing the samples (which is often impractical), or by using a different resuspension buffer that stimulates the stability of the sandwich complex of the antibody. Any buffer that stimulates stability and provides an accurate measurement of the detection antibody signal can be used to resuspend the magnetic spheres. It has been determined that TTBN-100 and DPBS meet these objectives, with DPBS being the most preferred resuspension buffer herein. The electrochemiluminescent Origin ™ technology uses an extremely stable ruthenium metal chelate that participates in a luminescent reaction in the presence of tripropylamine (TPA) after the application of an electrical potential. The paramagnetic spheres act as the solid phase and facilitate rapid test kinetics. The complex / spheres is transferred through a flow cell and captured magnetically on an electrode. Then a voltage is applied and the luminescence level is measured. The origin system ™ allows the formation of antigen-antibody complexes in a liquid phase. This method allows faster binding kinetics, and therefore shorter incubation times. With this method, the washing steps characteristic of the solid phase ELISA assays are significantly reduced. A surprising result of the use of the Origin ™ system and the various improvements in methodology developed during use (and described and claimed herein) is an improvement in the detection of all forms of type II collagen peptides. There is a natural variation in the peptides of type II collagen at position 774. Within the same individual, some type II collagen molecules contain a proline at position 774, while the rest of type II collagen contains a residue hydroxyproline in this position due to a post-translational modification. The above assay methods based on ELISA did not detect these different peptides with equal efficacy. However, the assay as described in the present example acts with equal efficacy on any of the peptides. This same equivalence in detection can also be obtained using proximity assays, in which labeled 9A4 is used as detection antibody. The assay that is preferred herein is as follows. Each control or test sample was analyzed directly or diluted in control urine to produce the final desired concentration of analyte. 50 μl of each test or control sample was combined with 25 μl of antibody mixture, and incubated at room temperature for one hour. The antibody mixture was formed by ruthenized 9A4 at 20 μg / ml and 5109 biotinylated at 10 μg / ml in TTBN-100. 25 μl of magnetic streptavidin spheres were added at 600 μg / ml in TTBN-100, and incubation was continued for 20 minutes. A magnet was used to hold the spheres against the sides of the container, and a pipette was used to remove essentially all of the fluid. The spheres were resuspended in 300 μl of DPBS, and the results were determined using the IGEN Origin ™ system, according to the manufacturer's instructions. Although this method produces reliable and highly reproducible measurements, it is expected that any sandwich immunoassay technique that does not require thorough washing steps can be developed to produce similar results, using this pair of binding and detection antibodies (5109 and 9A4). The washing steps need to be minimized because the monoclonal antibody 9A4 is a low affinity binder (1.7 x 10"7 M.) To increase the transfer and automate the assay, the assay is being modified for use in the Advantage System (Nichols, San Juan Capistrano, CA) In this assay modality, antibody 5109 is still biotinylated, although the detection antibody, 9A4, is labeled with acridinium ester.The specimens are incubated simultaneously with both antibodies.After an incubation period Initially, streptavidin-coated magnetic particles are added to the reaction mixture, followed by a second incubation.The acridinium ester is activated to emit light by the addition of hydrogen peroxide and an alkaline solution.This treatment causes oxidation of the product; the subsequent return to the ground state results in the emission of light that is quantified in 2 seconds and expressed in relative light units (RLU) by a luminometer of the system. The amount of bound marker is directly proportional to the concentration of type II collagen. Similarly, a TUNE assay can be developed using proximity scintillation, in which mAb 5109 binds to SPA spheres and mAb 9A4 is labeled with [125 I], the TUNE being determined by counting in a scintillation counter B. a TUNE assay can be imagined using any number of enzymatic or radioactive colorimetric markers.

EXAMPLE 10 Measurement of TUNE fragments in patients with osteoarthritis Using an assay similar to that described in Example 8, urinary levels of TUNE fragments were measured in patients with osteoarthritis and control patients (Table 13). It was shown that high levels of TUNE fragments were found in patients with osteoarthritis, when compared to controls. The inter-patient variability of TUNE levels over time in individuals with stable disease obtained in individual urine specimens was approximately 25%. Elevated levels of TUNE in patients with osteoarthritis were associated with more severe disease, as indicated by radiological tests of joint space narrowing and Kellgren's score (Table 14).

TABLE 13 Comparison of TUNE levels in normal subjects and OA Distribution of type II collagen Controls OA controls (less than 40) (same age) N 20 33 126 Interval 256-916 250-4845 773-9700 Medium 449 1066 2006 Medium 336 900 1609 Value P < 0.048- - > / < • -O.0001 > < O.0001 - > TABLE 14 Comparison between TUNE values and OA indices Correlation with the X-rays of the baseline First sample Second sample (N = 47) (N = 33) Likert score R = 0.40 (p = 0.006) R = 0.30 (p = 0.087) Non-aligned knees R = 0.25 (p = 0.90) R = 0.24 (p = 0.176) Kellgren score R = 0.30 (p = 0.0390) R = 0.38 (p = 0.29) In other studies, immunohistochemistry was performed on cartilage samples from patients with osteoarthritis and control patients using the 9A4 antibody. It was shown that the cartilage of patients with osteoarthritis was stained with 9A4, while the cartilage of the control patients did not. This indicates that there is not a significant amount of neoepitope exposed in the healthy cartilage, while the diseased cartilage contains a significant amount of exposed neoepitope. The hypothesis is that it is the result of degradation by collagenase in patients with osteoarthritis. In all protein detection assays, it must be determined that the species detected is indeed the protein of interest. The specificity of the urinary TUNE fragments present in the samples used in this example was confirmed by purifying the protein from the samples through an affinity column for 5109, followed by a mass spectrometric analysis of the fractions (Figure 4).

EXAMPLE 11 Measurement of TUNE fragments in patients with rheumatoid arthritis Monoclonal antibodies 9A4 and 5109 were used in an electrochemiluminescent assay to allow specific detection of the type II collagen neoepitope disruption by MMP-1, MMP-8 and MMP-13. Urinary TUNE levels were controlled in studies with patients with rheumatoid arthritis and control individuals. Sequential specimens from more than 150 individuals with moderate RA were analyzed, with baseline, with the results of the 6-month and 2-year X-rays of many of the subjects being available. The results of retrospective analyzes of urine samples from patients treated with methotrexate or corticosteroids were compared with those who were subjected to only conventional NSAID therapy (Figure 5). The median TUNE levels in patients with rheumatoid arthritis was almost double that observed in the control population, with a variability in individual urinary TUNE levels of approximately 25% (Figure 6). The median TUNE levels observed in individuals undergoing treatment with methotrexate were 30% lower than those receiving NSAIDs. The TUNE values correlate with various measures of the disease status of rheumatoid arthritis, including swollen and painful joint counts and the overall evaluation of the physician. In addition, when the TUNE levels were compared with the X-ray scores, it was observed that this marker correlates with a worsening of the disease state and predicts it (table 15). These results indicate that this marker is associated with the stage and pathogenesis of the disease, and can reasonably be expected to act as a prognosis for the outcome of the disease.TABLE 15 TUNE levels correlate with measures of disease severity in short-term and long-term RA studies.

Inverse correlation of methotrexate with the TUNE level (p = 0.012) Yalores p < 0.05 High collagen scores predict high X-ray scores and positive (worsening) changes in X-ray scores.

LIST OF SEQUENCES < 110 > Saltarelli, Mary J. Johnson, Kimberly S. Ottemess, Ivan G. < 120 > Assays for the measurement of type II collagen fragments in urine < 130 > PC9946A CIP < 140 > < 141 > < 160 > 70 < 170 > Patentln version 2.0 < 210 > 1 < 211 > 7 < 212 > PRT < 213 > Homo sapiens < 400 > 1 Gly Pro Pro Gly Pro Gln Gly 1 5 < 210 > 2 < 211 > 7 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > RES_MOD < 222 > (3) < 223 > fragment of type II collagen with 4Hyp < 400 > 2 Gly Pro Xaa Gly Pro Gln Gly 1 5 < 210 > 3 < 211 > 10 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > DOMAIN < 222 > (1) .. (10) < 223 > fragment of type 11 collagen < 400 > 3 Gly Glu Pro Gly Asp Asp Gly Pro Ser 1 5 10 < 210 > 4 < 211 > 10 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > RES_MOD < 222 > (3) < 223 > fragment of type II collagen with 4Hyp < 400 > 4 Gly Glu Xaa Gly Asp Asp Gly Pro Ser Gly 1 '5 10 < 210 > 5 < 211 > 408 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > signal peptide < 222 > (1) .. (33) < 223 > corresponds to a portion of the signal peptide of the heavy chain of 9A4 < 220 > < 221 > V region < 222 > (34) .. (378) < 223 > heavy chain sequence of mature 9A4 after cleavage of the signal peptide < 220 > < 221 > region C < 222 > (379) .. (408) < 223 > portion of the CH1 sequence of murine IgG1 < 400 > 5 ttcctgatgg cagctgccca aagtatccaa gcacagatcc agttggtgca gtctggtcct 60 agcctggaga gagctgaaga gacagtcaag atctcctgca aggcttctgg ttataccttc 120 acagactatt caatacactg ggtgaagcag gctccaggaa agggtttaaa gtggatgggc 180 tggataaaca ctgagactgg tgagccaaca tatgcagatg act caaggg acggtttgcc 240 ttctctttgg aaacctctgc cagcactgcc tatttgcaga tcaacaacct caaaaatgag 300 gacacggcta catatttctg tgctaggggc ggtagccttg actactgggg ccaaggcacc 360 actctcacag tctcctcagc caaaacgaca tctatcca cccccatctg 408 < 210 > 6 < 211 > 359 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > signal peptide < 222 > (1) .. (23) < 223 > signal peptide portion of the VL of 9A4 < 220 > < 221 > V region < 222 > (24) .. (341) < 223 > VL of mature 9A4 < 220 > < 221 > region C < 222 > (342) .. (359) < 223 > portion of the constant region kappa C muri ina < 400 > 6 actgtccaga cctcagtcat ttctcaccca ggacaaattg gtctccagta catctccagg ttcatgtctg 60 ggagaaggtc accatgacct gcagtgccag ctcaagtgta agttacatgt 120 gcagaagcca actggtacca ggatcctccc ccagactcct gattsatgcs tggcttctgg acatccaacc 180 agtccctgtt cgcttcagtg gcggtgggtc tgggacctct tactctctca 240 caatcagccg aatggaggct gaagatgctg ccacttatta ctgtcagcag tggagaagtt 300 atacacggac gttcggtgga ggcaccaagc tggaaatcat acgggctgat gctgcacca 359 < 210 > 7 < 211 > 883 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: single chain antibody 9A4, VH - VL. < 220 > < 221 > signal peptide < 222 > (29) .. (94) < 223 > Signal peptide introduced by engineering in pCANTAB6; the initiation methionine is probably encoded by the gtg codon. < 220 > < 221 > Mature peptide < 222 > (95) .. (880) < 223 > coding sequence of the single chain antibody produced by genetic engineering - 9A4 VH - VL. < 400 > 7 aagctttgga gccttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt 60 tgttcctttt tatgcggccc agccggccat ggcccagats sagttggtgs agtctggtcc 120 tgagc Gaag aagcctggag agacagtcaa gatctcctgc aaggcttctg gttatacctt 180 cacagactat tcaatacact gggtgaagca aagggtttaa ggctccagga agtggatggg 240 ctggataaac actgagactg gtgagccaac atatgcagat gacttcaagg gacggtttgc 300 cttctctttg gaaacctctg ccagsactgc ctatttgcag atcaacaacc tcaaaaatga 360 ggacacggct acatatttct gtgctagggg cggtagcctt gactactggg gccaaggcac 420 cactctcaca gtctcctcag gtggaggcgg ttcaggcgga ggtggcagcg gcggtggcgg 480 atcgcaaatt gttctcaccc agtctccagt attcatgtct gcatctccag gggagaaggt 540 caccatgacc tgcagtgcca gctcaagtgt aagttacatg tactggtacc agcagaagcc 600 aggatcctcc cccagactcc tgattcatgc cacatccaac ctggcttctg gagtccctgt 660 tcgcttcagt ggcggtgggt ctgggacctc ttactctctc acaatcagcc gaatggaggc 720 tgaagatgct gccacttatt actgtcagca gtggagaagt tatacacgga cgttcggtgg 780 aggcaccaag ctggaaatca tagcggccgc acatcatcat caccatcacg gggccgcaga 840 acaaaaactc atctcagaag aggatctgaa tggggccgca tag 883 <; 210 > 8 < 211 > 1340 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: single chain antibody 9A4 VL - VH. < 220 > < 221 > signal peptide < 222 > (293) .. (358) < 223 > signal peptide B in pATDFLAG. < 220 > < 221 > Mature peptide < 222 > (359) .. (1 126) < 223 > coding sequence of the single chain antibody produced by genetic engineering - 9A4 VL - VH. < 400 > 8 ctcatgtttg acagcttatc atcgatgaat tccatcactt ccctccgttc atttgtcccc 60 ggtggaaacg aggtcatcat ttccttccga aaaaacggtt gcatttaaat cttacatata 120 aaagactaca taatactttc tttgtaagat ttgatgtttg ag cggctga aagatcgtac 180 gtaccaatta ttgtttcgtg attgttcaag ccataacact gtagggatag tggaaagagt 240 gcttcatctg gttacgatca atcaaatatt caaacggagg gagacgattt tgatgaaata 300 cctattgcct acggcagccg ctggattgtt attactcgct gcccaaccag ccatggccca 360 aattgttctc acccagtctc cagtattcat gtctgcatct ccaggggaga aggtcascat 420 gacctgcagt gccagctcaa gtgtaagtta catgtactgg taccagcaga agccaggatc 480 ctcccccaga ctcctgattc atgccacatc caacctggct tctggagtcc ctgttcgctt 540 gggtctggga cagtggcggt cctcttacts tctcacaatc agccgaatgg aggctgaaga '600 tgctgccact tattactgtc agcagtggag aagttataca cggacgttcg gtggaggcac 660 atcatactta caagctggaa gtgcggacga tgcgaaaaag gatgctgcga agaaggatga 720 gacgatgcta cgctaagaaa aaaaggacct cgagatccag ttggtgcagt ctggtcctga 780 gctgaagaag cctggagaga cagtcaagat ctcctgcaag gcttctggtt ataccttcac 840 agactattca attacks ctggg tgaagcaggc tccaggaaag ggtttaaagt ggatgggctg 900 gataaacact gagactggtg agccaacata tgcagatgac ttcaagggac ggtttgcctt 960 acctctgcca ctctttggaa gcactgccta tttgcagatc aacaacctca aaaatgagga 1020 cacggctaca tatttctgtg ctaggggcgg tagccttgac tactggggcc aaggcaccac 1080 tcctcagcta tctcacagtc ggacgatgat gcgactacaa gacaaataaa aacctagcga 1140 tgaatccgtc aaaacatcat cttacataaa gtcacttggt gatcaagctc atatcattgt 1200 ccggcaatgg tgtgggcttt ttttgttttc tatctttaaa gatcatgtga agaaaaacgg 1260 ctgcgggaaa gaaaatcggt ggaccgggtt tttgtcgaaa tcataggcga atgggttgga 1320 ttgtgacaaa attcggatcc 1340 < 210 > 9 < 211 > 907 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: single chain antibody 5109 VH - VL < 220 > < 221 > signal peptide < 222 > (29) .. (94) < 223 > Signal peptide introduced by engineering into pCAN β; the initiation methionine is probably encoded by the gtg codon. < 220 > < 221 > Mature peptide < 222 > (95) .. (895) < 223 > coding sequence of the single chain antibody produced by genetic engineering - 5109 VH - VL. < 400 > 9 aagctttgga gccttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt 60 tgttcctttt tatgcggccc agccggccat ggccgaagtg cagctggtgg agtctggggg 120 aggctcagtg cagcctggag ggtccctgaa actctcctgt gcagcctctg gattcacttt 180 caatacctac ggcatgtctt gggttcgcca gactccagac aagaggctgg agtgggtcgc 240 aaccattaat agtaatggtg gtctcacctt ttatgcagac agtgtgaagg gccgattcac 300 gacaatgcca catttccaga aaaacaccct gtatctgcaa atgaacaggc tgaagtctgg 360 ggactcaggc atgtattact gtgtaagagg atatagtaat tacgctcgct ggggccaagg 420 ggcgctggtc actgtctcga gtggtggagg cggttcaggc ggaggtggca gcggcggtgg 480 gatgttgtga cggatcgtct tgacccaaac tccactcact ttgtcggtta ccattggaca 540 atctcttgca atcagcctcc agtcaagtca gagcctctta gattgacata ggtagtgatg 600 ttgttgcaga tttgatttgg ggccaggcca gtctccaaag cgcctaatct ttctggtgtc 660 tgaattggac tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac 720 actgaaaatc agcagagcgg aggctgaaga tttgggagtt tattattgct gccaaggtac 780 acattttcct cacacgttcg gtgctgggac caagctggag ctgaaagcgg ccgcagaact 840 aaaactcatc tcagaagagg atctgaatgg ggccgcacat caccaccatc accattaata 900 agaattc 907 < 210 > 10 < 211 > 348 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > V region < 222 > (1) .. (348) < 223 > VH region of 5109 ripe < 400 > 10 gaagtgcagc tggtggagtc tgggggaggc tcagtgcagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcaat acctacggca tgtcttgggt tcgccagact 120 ccagacaaga ggctggagtg ggtcgcaacc attaatagta atggtggtct caccttttat 180 gcagacagtg tgaagggccg attcaccatt tccagagaca atgccaaaaa caccctgtat 240 acaggctgaa ctgcaaatga gtctggggac tcaggcatgt attactgtgt aagaggatat 300 agtaattacg ctcgctgggg ccaaggggcg ctggtcactg tctctgca 348 < 21O > 11 < 211 > 336 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > V region < 222 > (1) .. (336) < 223 > VL region of mature 5109 < 400 > 11 gatgttgtga tgacccaaac tccactcact ttgtcggtta ccattggaca atcagcctcc 60 agtcaagtca atctcttgca gagcctctta ggtagtgatg gattgacata tttgatttgg 120 ggccaggcca ttgttgcaga gtctccaaag cgcctaatct ttctggtgtc tgaattggac 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgaaga tttgggagtt tattattgct gccaaggtac acattttcct 300 cacacgttcg gtgctgggac caagctggag ctgaaa 336 < 210 > 12 < 211 > 13 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > RES_MOD < 222 > (2) < 223 > fragment of type II collagen with 4Hyp < 220 > < 221 > RES_MOD < 222 > (8) < 223 > fragment of type II collagen with 4Hyp < 220 > < 221 > RES_MOD < 222 > (11) < 223 > fragment of type II collagen with 4Hyp < 400 > 12 Ala Xaa Gly Glu Asp Gly Arg Xaa Gly Pro Xaa Gly Pro * 1 5 10 < 210 > 13 < 211 > 20 < 212 > PRT < 213 > Homo sapiens «220 > < 221 > RES_MON < 222 > (9) < 223 > fragment of type II collagen with 4Hyp «220 > «220 > < 221 > RES_MOD < 222 > (15) < 223 > fragment of type II collagen with 4Hyp 220 > < 221 > RES_MOD < 222 > (18) < 223 > fragment of type II collagen with 4Hyp < 400 > 13 Gly Lys Val Gly Pro Ser Gly Ala Xaa Gly Glu Asp Gly Arg Xaa Gly 1 5 10 15 Pro Xaa Gly Pro 20 < 210 > 14 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > DOMAIN < 222 > (1) .. (9) < 223 > fragment of type II collagen < 400 > 14 Wing Glu Gly Pro Pro Gly Pro Gln Gly 1 5 < 210 > 15 < 211 > 10 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > RES_MOD < 222 > (3) < 223 > fragment of type II collagen CON 4Hyp < 220 > 221 > SITE < 222 > (7) < 223 > collagenase breaks at the end -COOH of the rest < 400 > 15 Gly Pro Xaa Gly Pro Gln Gly Leu Ala Gly 1 5 10 < 210 > 16 < 211 > (7) < 212 > PRT < 213 > Homo sapiens «220 > < 221 > DOMAIN < 222 > (1) .. (7) < 223 > Type II collagen fragment < 400 > 16 Gly Thr Pro Gly Pro Gln Gly 1 5 < 210 > 17 < 211 > 10 < 212 > PRT < 213 > Artificial sequence < 220 > < 223 > description of the artificial sequence: fragment of type collagen Modified II < 400 > 17 Cys Ala Glu Gly Pro Pro Gly Pro Gln Gly 1 5? < 210 > 18 < 211 > 10 < 212 > PRT < 213 > artificial sequence < 220 > < 223 > description of the artificial sequence: modified type II collagen fragment < 400 > 18 Cys Gly Glu Pro Gly Asp Asp Gly Pro Ser 1 5. 10 < 21O > 19 < 211 > 9 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > DOMAIN < 222 > (1) ... (9) < 223 > fragment of type II collagen < 400 > 19 Gly Glu Pro Gly Asp Asp Gly Pro Ser 1 5 < 210 > 20 < 211 > 19 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > DOMAIN < 222 > (1) ... (19) < 223 > fragment of type II collagen < 400 > 20 Gly Glu Pro Gly Asp Asp Gly Pro Be Gly Wing Glu Gly Pro Pro Gly 1 5 10 15 Pro Gln Gly < 210 > 21 < 211 > 20 < 212 > DNA < 213 > Mus musculus < 400 > 21 ggtgaagttg atgtcttgtg 20 < 210 > 22 < 211 > 23 < 212 > DNA < 213 > Mus musculus < 400 > 22 gaccttgcat ttgaactcct tgc 23 < 210 > 23 < 211 > 20 < 212 > DNA < 213 > Mus musculus < 400 > 2. 3 ggctgtggaa cttgctattc 20 < 210 > 24 < 211 > 20 < 212 > DNA < 213 > Mus musculus < 400 > 24 gggtgtggac cttgccattc 20 < 210 > 25 < 211 > 20 < 212 > DNA < 213 > Mus musculus < 400 > 25 gggtgtggac cttgctattc 20 < 210 > 26 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: mixture of a series of oligonucleotides as PCR primers for the murine VH signal peptide region < 400 > 26 ggstgtggam cttgcyattc 20 < 210 > 27 < 211 > 18 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: mixing a series of oligonucleotides as PCR primers for the murine VL signal peptide region < 400 > 27 gcttcctgct aatca tg < 210 > 28 < 211 > 21 < 212 > DNA < 213 > Mus musculus < 400 > 28 ggcagcagat ccaggggcca g 21 < 210 > 29 < 211 > 33 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > description of the artificial sequence: primer of the Kappa region of the murine light chain with a Hind lll < 400 > 29 gggaaagctt gttaactgct cactggatgg tgg 33 < 210 > 30 < 211 > 22 < 212 > DNA < 213 > Erwinia carotovora < 400 > 30 ctattgccta cggcagccgc tg 22 < 210 > 31 < 211 > 20 < 212 > DNA < 213 > Bacillus licheniformis < 400 > 31 cacatgatct ttaaagatag 20 < 210 > 32 < 211 > 136 < 212 > PRT < 213 > Mus musculus < 220 > < 221 > SIGNAL < 222 > (1) .. (11) < 223 > VH signal peptide portion of 9A4 < 220 > < 221 > DOMAIN < 222 > (12) .. (126) < 223 > Mature 9A4 VH < 220 > < 221 > DOMAIN < 222 > (127) ... (136) < 223 > CH1 constant domain portion of murine lgG1 < 400 > 32 Phe Leu Met Ala Ala Ala Gln Ser lie Gln Ala Gln lie Glp Leu Val 1 5 10 15 Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys lie Ser 20 25 30 Cys Lys Wing Ser Gly Tyr Thr Phe Thr Asp Tyr Ser lie His Trp Val 35 40 45 Lys Gln Wing Pro Gly Lys Gly Leu Lys Trp Met Gly Trp lie Asn Thr 50 55 60 Glu Thr Gly Glu Pro Thr Tyr Wing Asp Asp Phe Lys Gly Arg Phe Wing 65 70 75 BO Phe Ser Leu Glu Thr Be Ala Be Thr Wing Tyr Leu Gln lie Aen Aßn 85 90 95 Leu Lys Aßn Glu Aap Thr Ala Thr Tyr Phe Cys Ala. Arg Gly Gly Ser 100 105 10 Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Being Wing Lys 115 120 125 Thr Thr Pro Pro Ser Val Tyr Pro 130 X35 < 210 > 33 < 211 > 119 < 212 > PRT < 213 > Mus musculus < 200 > < 221 > SIGNAL < 222 > (1 ) . . (7) < 223 > VL signal peptide portion of 9A4 < 200 > < 221 > DOMAIN < 222 > (8) .. (113) < 223 > VL of mature 9A4 < 200 > < 221 > DOMAIN < 222 > (114). . (119) < 223 > constant domain portion kappa C murine < 400 > 33 Ser Val He Leu Ser Arg Gly Glp He Val Leu Thr Gln Ser Pro Va 1 5 10 15 Phe Met Ser Wing Pro Pro Gly Glu Lys Val Thr Met Thr Cys Ser Al 20 25 30 Ser Ser Val Val Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly Se 35 40 45 Ser Pro Arg Leu Leu He His Wing Thr Ser Asn Leu Wing Ser Gly Va 50 55 60 Pro Val Arg Phe Ser Gly Gly Gly Ser Gly Thr Ser Tyr Ser Leu Th 65 70 7S 8 Be Arg Met Glu Wing Glu Asp Wing Wing Thr Tyr Tyr Cys Gln Gl 85 90 95 Trp Arg Ser Tyr Thr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu II 100 105 110 He Arg Wing Asp Ala Wing Pro 115 < 210 > 34 < 211 > 47 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR primer -SOE for the VH region of 9A4 5 ', which includes a restriction endonuclease site Sfí I < 400 > 3. 4 gaggaggccc agccggccat ggcccagatc cagttggtgc agtctgg 47 < 210 > 35 < 211 > 61 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR primer -SOE for the VH region of 9A4 3 ', which includes a scFv linker segment for the PCR reaction of assembly with VL of 9A4 < 400 > 35 gccgctgcca cctccgcctg aaccgcctcc accactcgag actgtgagag tggtgcctg 60 61 < 210 > 36 < 211 > 56 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR primer -SOE for the VL region of 5 '9A4, which includes an overlap in the scFv < 400 > 36 ggttcaggcg gaggtggcag cggcggtggc ggatcgcaaa ttgttctcac ccagtc 5 < 210 > 37 < 211 > 38 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR primer -SOE for the VL region of 9A4 3 ', which includes a restriction endonuclease site Not I < 400 > 37 gaaggacgcc ggcgtatgat ttccagcttg gtgcctcc < 210 > 38 < 211 > 36 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR primer -SOE for scFv 9A4, which includes a restriction endonuclease site Not < 400 > 38 aagaagcggc cgctatgatt tccagcttgg tgcctc 36 < 210 > 39 < 211 > 23 < 212 > DNA < 213 > Escherichia coli < 400 > 39 agcggataac aatttcacac'agg 23 < 210 > 40 < 211 > 284 < 212 > PRT < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: VH VI of 9A4 scFv < 220 > < 221 > SIGNAL < 222 > (1 ) . . (22) < 223 > pCANTAB6 signal peptide; the Val in position 1 is most likely the Met initiator. < 220 > < 221 > DOMAIN < 221 > (2. 3) . . (137) < 223 > VH domain of 9A4 < 220 > < 221 > DOMAIN < 222 > (138). . (152) < 223 > 15 amino acid connector < 220 > < 221 > DOMAIN < 222 > (153). . (258) < 223 > VL domain of 9A4 < 220 > < 221 > SITE < 222 > (262). . (267) < 223 > His brand < 220 > < 221 > SITE < 222 > (271). . (280) < 223 > brand myc < 400 > 40 Met Lys Lyß Leu Leu Phe Ala He Pro Leu Val Val Pro Phe Tyr Ala 1 5 10 15 Wing Gln Pro Wing Met Wing Gln He Gln Leu Val Gln Ser Gly Pro Glu 20 25 30 Leu Lys Lys Pro Gly Glu Thr Val Lys He Ser Cys Lyß Wing Ser Gly 35 40 45 Tyr Thr Phe Thr Asp Tyr Ser He His Trp Val Lys Gln Ala Pro Gly 50 55 60. Lys Gly Leu Lys Trp Met Gly Trp He Asn Thr Glu Thr Gly Glu Pro 65 70 75 80 Thr Tyr Wing Asp Afip Phe Lys Gly Arg Phe Wing Phe Ser Leu Glu Thr 85 90 9S Be Ala Be Thr Wing Tyr Leu Gln He Asn Asn Leu Lys Asn Glu Asp 100 105 110 Thr Ala Thr Tyr Phe Cys Wing Arg Gly Gly Ser Leu Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gln He Val Leu Thr Gln Ser Pro 145 150 155 160 Val Phe Het Ser Wing Pro Gly Glu Lys Val Thr Met Thr Cys Ser 165 170 175 Wing Being Ser Val Val Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly ISO 185 190 Being Pro Pro Arg Leu Leu He Hie Wing Thr Ser Aen Leu Wing Ser aly 195 200 205 Val Pro Val Arg Phe Ser sly Gly Gly Ser Gly Thr Ser Tyr Ser Leu 210 215 220 Thr lie Ser Arg Met Glu Ala slu Asp Ala Ala Thr Tyr Tyr Cys Gln 225 230 235 240 Gln Trp Arg Ser Tyr Thr - Arg Thr Phe Gly Gly Gly Thr Lys Leu Olu 245 250 255 He He Ala Ala Ala His His His His Hie His Gly Ala Ala Glu Gln 260 265 270 Lys Leu He Ser slu Glu Asp Leu Asn. Gly Ala Ala 275 280 < 210 > 41 < 211 > 21 < 212 > DNA < 213 > bacteriophage fd < 400 > 41 gtcgtctttc cagacgttag t 21 < 210 > 42 < 211 > 25 < 212 > PRT < 213 > artificial sequence < 220 > • * A I < 223 > Description of the artificial sequence: single chain antibody linker sequence < 400 > 42 Leu Ser Wing Asp Asp Wing Lys Lys Asp Wing Wing Lys Lys Asp Asp Wing 1 5 10 15 Lys Lys Asp Asp Ala Lys Lys Asp Leu 20 25 < 210 > 43 < 211 > 34 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR-SOE primer for the VL region of 5 '9A4, which includes a restriction endonuclease site Neo I < 400 > 43 gaggagccat ggcccaaatt gttctcaccc agtc 34 < 210 > 44 < 211 > 79 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR-SOE primer for the VL region of 9A4 3 ', which includes a scFv linker segment for the PCR reaction of assembly with VH of 9A4 < 400 > 44 ctttcttagc gtcatccttc ttcgcagcat cctttttcgc atcgtccgca ctaagcttgadO tttccagctt ggtgcctcc 7 < 210 > 45 < 211 > 70 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR-SOE primer for the VH region of 9A4 5 ', which includes a scFv linker segment for the PCR reaction of assembly with VL of 9A4 < 400 > 45 ctgcgaagaa ggatgacgct aagaaagacg atgctaaaaa ggacctcgag atccagttgg tgcagtctgg < 210 > 46 < 211 > 36 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR-SOE primer for the VH region of 9A4 3 ', which includes a restriction endonuclease site Nhe I < 400 > 46 gaggaagcta gctgaggaga ctgtgagagt ggtgcc < 210 > 47 < 211 > 278 < 212 > PRT < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: VL-VH of 9A4 scFv < 220 > < 221 > SIGNAL < 222 > (1 ) . . (22) < 223 > signal peptide B in pATDFLAG < 220 > < 221 > DOMAIN < 222 > (23) .. (128) < 223 > VL domain of 9A4 < 220 > < 221 > DOMAIN < 222 > (129) .. (153) < 223 > 25 amino acid connector < 220 > < 221 > DOMAIN < 222 > (154) .. (268) < 223 > VH domain of 9A4 < 220 > < 221 > SITE < 222 > (271) .. (278) < 223 > FLAG brand < 400 > 47 Met Lys Tyr in Leu Pro Thr Ala Wing Wing Gly Leu Leu Leu Leu Wing 1 5 10 15 Wing Gln Pro Wing Met Wing Gln He Val Leu Thr Gln Ser Pro Val Phe 20 25 30 Met Ser Wing Pro Pro Gly Glu Lys Val Thr Met Thr Cys Ser Wing Ser 35 40 45 Ser Ser Val Ser Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Oly Ser Ser 50 55 60 Pro Arg Leu Leu lie His Wing Thr Ser Aan Leu Wing Ser Gly Val Pro 65 70 75 80 Val Arg Phe Ser Gly Gly Gly Ser Gly Thr Ser Tyr Ser Leu Thr He 85 90 95 Be Arg Met Glu Wing Glu Asp Wing Wing Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Arg Ser Tyr Thr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu He He 115 120 125 Leu Ser Wing Asp Asp Wing Lys Lys Asp Wing Wing Lys Lys Asp Asp Wing 130 135 140 Lys Lys Asp Asp Wing Lys Lys Asp Leu Glu He Gln Leu Val Gln Ser 145 150 155 160 Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys He Ser Cys Lye 165 170 175 Wing Ser Gly Tyr Thr Phe Thr Asp Tyr Ser He His Trp Val Lys Gln 180 185 190 Wing Pro Gly Lyß Gly Leu Lys Trp Met Gly Trp He Asn Thr Glu Thr 155 200 205 Gly Glu Pro Thr Tyr Wing Aßp Asp Phe Lys Gly Arg Phe Ala Phe be 210 215 220 Leu Glu Thr Ser Ala Be Thr Ala Tyr Leu Gln He Asn Asn Leu Lys 225 230 235 240 Asn Glu Asp Thr Wing Thr Tyr Phe Cys Wing Arg Gly Gly Ser Leu Asp 245 250 255 Tyr Trp Gly sln Gly Thr Thr Leu Tlir Val Ser Ser Wing Ser Asp Tyr 260 265 270 Lys Asp Asp Asp Asp Lys 275 < 210 > 48 < 211 > 116 < 212 > PRT < 213 > -ftflus musculus < 220 > < 221 > D01WNK > < 222 > (1) .. (1 6) < 223 > Mature 5109 VH < 400 > 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser al Gln Pro Gly Gly 1 5 10 15 Be Leu Lys Leu Be Cys Wing Wing Be Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Wing Thr He Asn Ser Asn Gly Gly Leu Thr Phe Tyr Wing Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Wing Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Aßn Arg Leu Lys Ser Gly Asp Ser Gly Met Tyr Tyr Cys 85 90 35 Val Arg Gly Tyr Ser Asn Tyr Ala Arg Trp Gly Gln Gly Ala Leu Val 100 1"? 0n5c- 110 Thr Val Ser Wing 115 <210> 49 <211> 112 <212 PRT <213 >Mus musculus <220> <221> DOMAIN <222> (1) .. (112) <223> VL 5109 mature <400> 49 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr He Gly 1 5 10 15 Gln Ser .Ala Ser lie Cys Lys Ser Ser Gln Ser Leu Leu Gly Ser 20 25 30 Asp Gly Leu Thr Tyr Leu lie Trp Leu Leu Gln Arg Pro Gly Gln Ser * 0 45 Pro Lys Arg Leu He Phe Leu Val Ser Glu Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Qly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80 Ser Arg Val Glu Wing Glu Asp Leu Gly Val Tyr Tyr Cys Cys Gln Gly 85 90 95 Thr His Phe Pro His Thr Phe Gly Wing Gly Thr Lys Leu Glu Leu Lys 100 105 110 < 220 > 50 < 221 > 24 < 222 > DNA < 223 > Mus musculus < 400 > 50 gaagtgcagc tggtggagtc tggg < 210 > 51 < 211 > 20 < 212 > DNA < 213 > Mus musculus < 400 > 51 gatgttgtga tgacccaaac 20 < 210 > 52 < 211 > 24 < 212 > DNA < 213 Mus musculus < 400 > 52 ctgatcagtc caactgttca ggac 24 < 210 > 53 < 211 > 16 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: oligonucleotide sequencer < 400 > 53 gtaaaacgac ggccag 16 < 210 > 54 < 211 > 17 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: oligonucleotide sequencer < 400 > 54 caggaaacag ctatgac 17 < 210 > 55 < 21 1 > 49 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR-SOE primer for the VH region of 5109 5 ', which includes a restriction endonuclease site Sfi I < 400 > 55 gaagagcggc ccagccggcc atggccgaag tgcagctggt ggagtctgg 49 < 210 > 56 < 211 > 74 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR-SOE primer for the VH region of 5109 3 ', which includes a scFv linker segment for the assembly PCR reaction with VL of 5109 < 400 > 56 ccgccgctgc ccgccgctgc cacctccgcc tgaaccgcct ccaccactcg agacagtgac cagcgcccct tggc < 210 > 57 < 211 > 66 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR-SOE primer for the VL region of 5109 5 ', which includes an overlap in the scFv < 400 > 57 ggttcaggcg gaggtggcag cggcggtggc ggatcgtctg atgttgtgat gacccaaact ccactc < 210 > 58 < 211 > 87 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR-SOE primer for a 5109 3 'VL region, Ser version, which includes a restriction endonuclease site Not l < 400 > 58 ggaaggagcg gccgctttca gctccagctt ggtcccagca ccgaacgtgt gaggaaaatg tgtaccttgg gagcaataat aaactcc < 210 > 59 < 211 > 36 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: PCR-SOE primer for the 5L 5109 VL region, Cys version, which includes a restriction endonuclease site Not I < 400 > 59 ggaaggagcg gccgctttca gctccagctt ggtagc < 210 > 60 < 211 > 21 < 212 > DNA < 213 > Homo sapiens < 400 > 60 ctcttctgag atgagttttt g 21 < 210 > 61 < 211 > 18 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > description of the artificial sequence: oligonucleotide sequencer.

Primers in the Gly4Ser connector region. < 400 > 61 gaggcggttc aggcggag 18 < 210 > 62 < 211 > 18 < 212 > DNA < 213 > artificial sequence < 220 > < 223 > description of the artificial sequence: oligonucleotide sequencer.

Primers in the Gly4Ser connector region. < 400 > 62 gatccgccac cgccgctg 18 < 210 > 63 < 211 > 289 < 212 > PRT < 213 > artificial sequence < 220 > < 223 > Description of the artificial sequence: VH-VL of 5109 scFv < 220 > < 221 > SIGNAL < 222 > (1) .. (22) < 223 > pCANTAB6 signal peptide; the Val in position 1 is most likely the Met initiator. < 220 > < 221 > DOMAIN < 222 > (23) .. (138) < 223 > VH domain 5109 < 220 > < 221 > DOMAIN < 222 > (139) .. (154) < 223 > 16 amino acid connector < 220 > < 221 > DOMAIN < 222 > (155) .. (266) < 223 > VL domain 5109 < 220 > < 221 > SITE < 222 > (270) .. (279) < 223 > brand myc < 220 > < 221 > SITE < 222 > (284) .. (289) < 223 > His mark < 400 > 63 Met Lys Lys Leu Leu Phe Wing He Pro Leu Val Val Pro Phe Tyr Wing 1 5 10 15 Wing Gln Pro Wing Met Wing Glu Val Gln Leu Val Glu Ser Gly Gly Gly 20 25 3rd Ser Val sln Pro sly Gly Ser Leu Lys Ser Cys Wing Wing Ser Gly 35 40 45 Phe Thr Phe Asn Thr Tyr Gly Met Ser Trp Val Arg Gln Thr Pro Asp 50 55 60 Lys Arg Leu Glu Trp Val Wing Thr He Asn Ser Asn Gly Gly Leu Thr 65 70 75 80 Phe Tyr Wing Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn 85 9o 35 Wing Lys Asn Thr Leu Tyr Leu sln Met Asn Arg Leu Lys Ser Gly Asp 100 105 110 Ser Gly Met Tyr Tyr Cys Val Arg Gly Tyr Ser Asn Tyr Ala Arg Trp 115 120 125 Gly Gln Gly Ala Leu Val Thr Val Ser Ser Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Val Val Met Thr Gln 145 150 155 160 Thr Pro Leu Thr Leu Ser Val Thr He Gly Gln Ser Wing Ser He Ser 165 170 175 Cys Lys Ser Ser Gln Ser Leu Leu Gly Ser Asp Gly Leu Thr Tyr Leu 180 185 190 He Trp Leu Leu Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu He Phe 195 200 205 Leu Val Ser Glu Leu Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser 210 215 220 Gly Ser Gly Thr Asp Phe Thr Leu Lys He Ser Arg Ala Glu Ala slu 225 230 235 240 Asp Leu sly Val Tyr Tyr Cys Cys Gln Gly Thr His Phe Pro His Thr 245 250 255 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ala Ala Ala Glu Gln Lys 260 265 270 u ^ ß.

Leu He Ser Glu Glu Asp Leu Asn Ala Ala Ala nxs axa HIB HIS HIS 275 280 285 His < 210 > 64 < 211 > 21 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > RES_MOD < 222 > (17) < 223 > fragment of type II collagen with 4Hyp < 400 > 64 Glu Lys Gly Glu Pro Gly Asp Asp Wing Pro Ser Gly Wing Glu Gly Pro 1 5 10 15- Xaa Gly Pro Gln Gly 20 < 210 > 65 < 211 > 62 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the Artificial Sequence: PCR Seeding Oligonucleotide < 220 > < 221 > misc_difference < 222 > (25) .. (51) < 223 > PCR seed seeding oligonucleotide of 9A4 scFv. Planting level - 10% with other nucleotides than those that appear in this segment. The sequence that appears represents the original. < 400 > 65 gactgtgaga gtggtgcctt ggccccagta gtcaaggcta ccgcccctag cacagaaata 60 tg 62 210 > 66 < 211 > 26 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > V region < 222 > (1) .. (26) < 223 > portion of the JH segment of the VH of 9A4 used as a PCR primer. < 400 > 66 gccaaggcac cactctcaca gtctcc 26 < 210 > 67 < 211 > 21 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > RES_MOD < 222 > (3) < 223 > fragment of type II collagen with 4Hyp < 220 > < 221 > RES_MOD < 222 > (6) < 223 > fragment of type II collagen with 4Hyp < 220 > < 221 > RES_MOD < 222 > (9) < 223) fragment of type II collagen with 4Hyp < 220 > < 221 > RES_MOD < 222 > (18) < 223 > fragment of type II collagen with 4Hyp < 400 > 67 Gly Pro Xaa Gly Pro Xaa Gly Lys Xaa Gly Asp Asp Gly Glu Ala Gly 1 5 10 15 Lys Xaa Gly Lys Wing 20 < 210 > 68 < 211 > 21 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > RES_MOD < 222 > (3) < 223 > fragment of type II collagen with 4Hyp < 210 > < 220 > < 221 > RES_MOD < 222 > (18) < 223 > fragment of type II collagen with 4Hyp < 220 > < 221 > RES_MOD < 222 > (21) < 223 > fragment of type II collagen with 4Hyp < 400 > 68 Gly Pro Xaa Gly Pro Arg Gly Arg Ser Gly Glu Thr Gly Pro Wing Gly 1 5 10 15 Pro Xaa Gly Asn Xaa < 210 > 69 20 < 211 > 22 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > RES_MOD < 222 > (3) < 223 > fragment of type II collagen with 4Hyp < 220 > < 221 > RES_MOD < 222 > (12) < 223 > fragment of type II collagen with 4Hyp < 220 > < 221 > RES_MOD < 222 > (15) < 223 > fragment of type II collagen with 4Hyp < 220 > < 221 > RES_MOD < 222 > (18) < 223 > fragment of type II collagen with 4Hyp < 400 > 69 Gly Ala Xaa Gly Pro Gln Gly Phe Gln Gly Asn Xaa Gly Glu Xaa Gly 1 '5 10 15 Glu Xaa Gly Val Ser Tyr 20 < 210 > 70 < 211 > 20 < 222 > PRT < 223 > Homo sapiens < 220 > < 221 > RES_MOD < 222 > (3) < 223 > fragment of type II collagen with 4Hyp < 220 > < 221 > RES_MOD < 222 > (12) < 223 > fragment of type II collagen with 4Hyp < 200 > < 221 > RES_MOD < 222 > (15) < 223 > fragment of type II collagen with 4Hyp < 220 > < 221 > RES_MOD < 222 > (18) < 223 > fragment of type II collagen with 4Hyp < 400 > 70 Gly Glu Pro Gly Asp Asp Wing Gly Pro Ser Gly Wing Glu Gly Pro Xaa 1 5 10 15 Gly Pro Gln Gly 20

Claims (37)

NOVELTY OF THE INVENTION CLAIMS

1. A method for controlling fragments of type II collagen in urine, and said method comprises: a) contacting said urine with a capture antibody, wherein said capture antibody binds specifically to the fragments of type II collagen, up to the substantial exclusion of any binding with type I or III collagen fragments; b) contacting said urine with a detection antibody, wherein said detection antibody binds specifically to collagenase fragments generated by collagenase; and c) detecting the amount of type II collagen fragments attached to said capture and detection antibodies.

2. A method according to claim 1, wherein said capture and detection antibodies are monoclonal antibodies.

3. A method according to claim 1, wherein said detection antibody is active against the sequences appearing in SEQ ID NO: 1 and 2.

4. A method according to claim 3, wherein said antibody of detection has a VH sequence that is at least 95% homologous with that appearing in SEQ ID NO: 32, and a VL sequence that is at least 95% homologous with that appearing in SEQ ID NO: 33.

5. - A method according to claim 3, wherein said detection antibody has the same CDRs as the VH sequence appearing in SEQ ID NO: 32, and the same CDRs as the sequence V appearing in SEQ ID NO: 33.

6 A method according to claim 1, wherein said capture antibody is active against the sequences appearing in SEQ ID NO: 3 and 4.

7. A method according to claim 6, wherein said capture antibody it has a VH sequence that is at least 95% homologous with that appearing in SEQ ID NO: 48, and a VL sequence that is at least 95% homologous with that appearing in SEQ ID NO: 49.

8.- A The method according to claim 6, wherein said capture antibody has the same CDRs as the sequence V appearing in SEQ ID NO: 48, and the same CDRs as the sequence V appearing in SEQ ID NO: 49.

9.- A method according to claim 1, wherein said contact steps a) and b) occur simultaneously.

10. A method according to claim 9, wherein after said contacting steps and before said detection step, said capture antibody is immobilized on a magnetic material.

11. A method according to claim 10, wherein said capture antibody is biotinylated, and said magnetic material is a magnetic streptavidin sphere.

12. - A method according to claim 10, in which a majority of the unbound materials are separated from said immobilized capture antibody.

13. A method according to claim 12, wherein said immobilized capture antibodies are resuspended in a buffered solution before said detection step c).

14. A method according to claim 1, wherein said contact steps a) and b) occur sequentially.

15. A method according to claim 14, wherein after said contact step a) and before said contact step b), said capture antibody is immobilized on a magnetic material.

16. A method according to claim 15, wherein said capture antibody is biotinylated, and said magnetic material is a sphere of magnetic streptavidin.

17. A method according to claim 15, in which a majority of the unbound materials are separated from said immobilized capture antibody.

18. A method according to claim 17, wherein said immobilized capture antibodies are resuspended in a buffered solution prior to said detection step c).

19. A method according to claim 1, wherein said capture antibody is present in said contact step a) at a concentration of from about 5 to about 20 μg / ml.

20. - A method according to claim 19, wherein said capture antibody is present in said contact step a) at a concentration of about 10 μg / ml.

21. A method according to claim 1, wherein said detection antibody is present in said contact step b) at a concentration of from about 10 to about 30 μg / ml.

22. A method according to claim 21, wherein said detection antibody is present in said contact step b) at a concentration of about 20 μg / ml.

23. A method according to claim 1, wherein said detection step is carried out by detecting the result of an enzymatic reaction, an electrochemical signal, an optical signal, a radioactive signal, a fluorescent signal or by a test proximity of scintillation (SPA).

24. A method according to claim 1, wherein said detection step is carried out by measuring the electrochemiluminescence, the light or luminescence emitted, and is read by a scintillation counter ß.

25. A method according to claim 1, wherein said contact steps a) and b) are carried out in a buffer comprising Tris pH 7.4 at a concentration between 50 and 250 mM, Tween-20 at a concentration between 0.5 and 2.0%, BSA at a concentration between 0.5 and 2.0%, and NaCl at a concentration between 100 and 200 mM.

26. - A method according to claim 1, comprising the additional steps of: d) contacting a series of control samples with said capture antibody; e) contacting said control samples with said detection antibody; and f) detecting the amount of type II collagen fragments in said control samples bound to said capture and detection antibodies.

27. A method according to claim 26, wherein said control samples comprise known amounts of type II collagen fragments diluted in control urine.

28. A method according to claim 1, comprising the additional step of removing unbound detection antibody after said contact step b) and before said detection step c).

29.- A test kit to control the urine to detect fragments of type II collagen, and said test kit comprises: a) a Capture antibody, wherein said capture antibody binds specifically to the collagen type II fragments, to the substantial exclusion of any binding with collagen fragments of type I or III; b) a detection antibody, wherein said detection antibody binds specifically to collagen fragments generated by collagenase; c) a 20 container; and d) instructions describing a method for using said first antibody and said second antibody to control a biological means for detecting fragments of type II collagen.

30. - An assay kit according to claim 29, wherein said detection antibody has a VH sequence that is at least 95% homologous to that appearing in SEQ ID NO: 32, and a VL sequence that is at least 95% homologous to that appearing in SEQ ID NO: 33, and said capture antibody has a sequence VH that is at least 95% homologous to that appearing in SEQ ID NO: 48, and a sequence V that is at least 9N / or homologous with that appearing in SEQ ID NO: 49.

31.- An assay kit according to claim 29, wherein said detection antibody has the same CDR as the sequence VH that appears in SEQ ID NO: 32 , and the same CDRs as the sequence VL that appears in SEQ ID NO: 33, and said capture antibody has the same CDR as the sequence VH that appears in SEQ ID NO: 48, and the same CDR as the sequence VL that appears in SEQ ID NO: 49.

32.- An assay kit according to claim 29, and said test kit further comprises: e) a control fluid. The positive, comprising control urine containing a known amount of collagen type II fragments; and f) a dilution fluid, comprising control urine to dilute the samples that are being tested with said test kit.

33.- A biodetector plate for detecting the presence of an immunological binding event, in which said plate comprises a first active antibody against the sequences appearing in SEQ ID NO: 1 or 2, or a second active antibody against the sequences that appear in SEQ ID NO: 3 or 4.

34. - A method for diagnosing a patient a disease state associated with the degradation of type II collagen, and said method comprises the step of detecting the presence of abnormally high amounts of type II collagen fragments in urine collected from said patient.

35. A method according to claim 34, wherein said disease eluate is osteoarthritis or rheumatoid arthritis.

36.- A method to carry out a clinical trial to evaluate a drug which is believed to be useful for treating a disease state associated with the degradation of type II collagen, and this method comprises: a) measuring the level of type II collagen fragments in urine collected from a series of patients; b) administering said drug to a first subset of said patients, and a placebo to a second subset of said patients; c) repeating step a) after the administration of said drug or said placebo; and d) determining whether said drug is reducing the amount of type II collagen fragments present in said urine of said first subset of patients, to a degree that is statistically significant when compared to any reduction occurring in said second subset of patients, in which a statistically significant reduction indicates that said drug is useful for treating said disease state.

37. A method according to claim 36, wherein said disease state is osteoarthritis or rheumatoid arthritis.