WO2005063335A2 - MONOCLONAL DES-LYS58-β2-MICROGLOBULIN ANTIBODIES FOR MEASUREMENT AND MANAGEMENT OF HEMODIALYSIS COMPLICATIONS - Google Patents

Abstract

β2-microglobulin (β2m) is susceptible to specific cleavage after and subsequent removal of its Lysine-58 amino acid residue in various chronic disease conditions. This has been shown by incubating patient sera with normal β2m and following structural alterations by electrophoretic techniques. At present, only very elaborate analytical methods can be used to analyze the state of β2m in a complex sample matrix such as a serum sample because no specific reagent, e.g. an antibody, is available. The present invention provides antibodies that are specific for the cleaved variant of β2m, des Lys58-β2-microglobulin, in free solution in biological fluids. These antibodies may be used in methods for detecting des-Lys58-β2-microglobulin and in particular in in vitro diagnostic procedures. In addition to such methods, the invention provides antibodies for use as medicaments and for the preparation of medicaments as well as a method for generating the antibodies, devices for reducing the content of des-Lys58-β2-microglobulin in a given material such as a hemodialysis fluid and, finally, in vitro diagnostic kits.

Description

Monoclonal des-Lys⁵⁸-β2-microglobulin antibodies for measurement and management of hemodialysis complications

FIELD OF INVENTION

The present invention relates to specific immunological reagents, such as IgG monoclonal antibodies, which specifically bind to des-Lys⁵⁸-β2-microglobulin while not cross-reacting with β2-microglobulin. One part of the invention is directed to the antibodies per se, while other parts of the invention pertains to methods for detecting, quantitating and monitoring des-Lys⁵⁸-β2-microglobulin. Such methods may be performed as diagnostic procedures for diagnosing an amyloid disease or for identifying subjects at risk for developing an amyloid disease. Still other parts of the invention provide pharmaceutical compositions and methods of treatment of amyloid diseases as well as use of the antibodies as medicaments and for the preparation of medicaments. Finally, some parts of the invention provide a device for reducing the amounts of des-Lys⁵⁸-β2-microglobulin in a biological material and in vitro diagnostic kits comprising the antibodies of the invention

GENERAL BACKGROUND

β₂-microglobulin (β₂m) is a small serum protein that is also found on the surface of most cells as the non-covalently associated light chain of the MHC-class I molecules. It circulates in plasma in low concentrations (0.9-2.5 μg/ml corresponding to 77-213 nM) and has no known functions in its free form. Structurally, the protein consists of 99 amino acid residues making up two disulfide-interconnected domains - that are also connected by the polypeptide backbone - of 3 and 4 β-strands, respectively and containing very little additional secondary structure. The three-dimensional structure of β₂m is known from crystallography and from NMR spectroscopy.

β₂m is one of about 20 different proteins that are know to be able to misfold in vivo leading to characteristic and destructive structures consisting of insoluble aggregated stacked protein or polypeptide β-sheets in organs and tissues. The aggregated material has specific staining properties and ultrastructure and is called amyloid and a disease associated with such deposits is generically referred to as an amyloidosis. Amyloid is the common end result of protein conformational diseases such as senile amyloidosis, Alzheimers's disease and prion diseases among others. β₂m amyloidosis is a complication of chronic kidney disease and long-term hemodialysis. The disorder is characterised by the development of severe complications such as compressed median nerve (carpal tunnel syndrome), joint pain, and joint swelling that are typical in this group of patients with dialysis-related amyloidosis (DRA). In DRA apparently normal β₂m assembles into insoluble fibrillar material consisting of protofilaments displaying the extended twisted cross β-sheet core structure that is characteristic of amyloid. Others and we have described soluble conformational intermediates of intact β₂m that can be induced by the presence of organic solvent and/or low pH. These alternatively folded species may represent stages on the early part of the amyloid misfolding pathway and may even exist as a minor fraction of normal β₂m. This unstability is common to many amyloid precursor proteins that may be induced to produce amyloid fibrils in vitro by slightly denaturing conditions. However, an increased concentration of β₂m is not enough by itself to initiate amyloid formation and there appears to be no simple relationship between the serum concentration of β₂m and the development and extent of β₂m amyloidosis. Thus, the precise mechanism leading to β₂m-amyloid formation in vivo is still unknown.

In chronic disease the circulating β₂m may be modified from the single-chain 99-amino acid polypeptide into a heterodimeric disulfide-linked 98 amino acid protein by the concerted action of activated complement and carboxypeptidase activities in serum. Even though direct evidence for this taking place in vivo has not yet been presented it has been shown that a specific β₂m-cleaving activity is greatly enhanced in inflammatory sera and sera from cancer patients. The modified β₂m is thought to arise from the action of activated complement Cls e.g. activated by immune complexes that cleaves β₂m C- terminally to Lys⁵⁸. In serum, this exposed lysine residue is rapidly removed by a carboxypeptidase B-like activity and the modified form of β₂m, des-Lys⁵⁸-β2-microglobulin (also denoted ΔK58-β₂m), is generated.

While the material recovered from the amyloid deposits of hemodialysis patients have been reported to consist of intact wild-type (wt) β₂m various modified forms have also been reported in the circulation of these patients and the purification methods from amyloid may not be able to recover altered β₂m. We have described the conformationally instability of des-Lys⁵⁸-β2-microglobulin and thus, this variant may be involved in the formation of amyloid in vivo (Heegaard et al 2002, JBC). Also, it has previously been shown that the β₂m-converting activity of sera from patients with rheumatoid arthritis, systemic lupus erythematosus, malignant lymphoma, and other cancers is increased as compared to the activity of serum from normal individuals. Thus, des-Lys⁵⁸-β2-microglobulin may be a marker of chronic disease and specifically of complications in in hemodialysis patients. The mechanisms behind the increased β₂m-converting activity in patient sera are unknown but in patients treated with hemodialysis it may be due to activation of the complement system by the dialysis procedure, e.g. by different types of dialysis membranes. It would be of obvious benefit to have an assay method for the biocompatibility of a dialysis procedure and an important marker for the positive or negative effects of new methods of treatments.

The measurement and immunoaffinity isolation of β₂-microglobulin (β₂m) in biological fluids is conventionally performed using both monoclonal and polyclonal antibodies that have been raised by immunization with normal, intact β₂m (REF e.g. Bjerrum OW, Birgens HS. Measurement of beta-2-microglobulin in serum and plasma by an enzyme-linked immunosorbent assay (ELISA). Clin Chim Acta 1986; 155: 69-76; Sen JW, Bergen HR, Heegaard NHH. On-line immunoaffinity-liquid chromatography-mass spectrometry for identification of amyloid disease markers in biological fluids. Anal Chem 2003; 75: 1196- 1202). Such antibodies will react both with β₂m and with des-Lys⁵⁸-β2-microglobulin (des- lys⁵⁸-β₂m) (Fig. l). Monoclonal antibodies that are specific for β₂m and which do not cross- react with des-Lys⁵⁸-β2-microglobulin have not yet been described. Tachibana et al (1984) describes a monoclonal antibody raised against β₂m which also reacts with des-Lys⁵⁸-β2- microglobulin. The monoclonal antibodies desribed in the present application react specifically with des-Lys⁵⁸-β2-microglobulin and do not cross-react with β₂m.

As des-lys⁵⁸-β₂-microglobulin is present in the circulation in much lower amounts compared to intact β₂m, sensitive and specific immunoassays as for instance capture or sandwich immunoassays will be preferred for reliable detection and quantification of this variant. In a capture immunoassay, an antibody is used as a capturing agent for β₂-microglobulin, including any structural variants present, in serum, plasma, other biofluids and tissue extracts. This enables the isolation of the β₂m molecular family from complicated sample matrices. To subsequently detect the desLys-β₂m molecule captured by the surface- immobilized antibody with no interference from other protein or peptide components present in the original sample, binding of a specific monoclonal antibody to the des-Lys⁵⁸- β₂m is required. The bound monoclonal antibody is then detected by secondary immunoreagents such as enzyme-, radioactivity-, fluorescein-, or other reporter group- labelled secondary antibody conjugates directed against mouse antibodies. In this assay procedure, the capture step has a concentrating effect on the molecular species to be detected, which results in increased sensitivity of the assay.

EPO0229723A(A2) describes the development of monoclonal IgM antibodies specific for des-lys⁵⁸-β₂m. Monoclonal IgM antibodies differ from IgG antibodies by being large molecules (pentamers instead of monomers) and are generally of lower affinity since they are involved in the primary immune response (e.g. : Goldsby RA, Kindt TJ, Osborne, BA, Kuby J. Immunology. Fifth Edition, W. H. Freeman & Co, New York 2003). Thus, for quantitative immunoassays IgM class monoclonal antibodies are normally not desired because of their low affinity, difficulty of purification from culture supernatants, and risk of steric interference with the other molecules used in a particular assay design. Specifically for the previously developed IgM antibodies against des-lys⁵⁸-β₂m described in EPO0229723A(A2) it was found that they required their target antigen, des-Lys⁵⁸-β₂m, was directly immobilised on a surface to be detectable (personal communication Mogens H. Nissen; inventor on EP229723)). Thus, these IgM class monoclonal antibodies rely on specific unfolding of the target antigen for proper display and subsequent binding to the epitope which is inaccessible for the large IgM antibodies in the natively folded des-lys⁵⁸- β₂m target molecules in solution. Since IgM anti-des-lys⁵⁸-β₂m binding is confined to situations where the des-lys⁵⁸-β₂m is immobilised on a surface it is difficult to use it for quantitative assays of des-lys⁵⁸-β₂m in complex biofluids such as blood, serum, plasma and cerebrospinal fluid. Also it is impossible to benefit from the concentrating effect of capture immunoassays using these antibodies. Accordingly, these antibodies have not, to our knowledge, been used for the development of any assays suitable for des-lys⁵⁸-β₂m detection and quantitation in biological fluids and therefore have limited potential use in the diagnosis, screening, monitoring, and treatment of amyloisidosis, including complications associated with chronic hemodialysis treatment (DRA).

EPO0236509 (Al) describes an apparatus and the use of an anti-β₂m antibody immobilised to a carrier to adsorb and remove β₂m from the blood in patient undergoing blood dialysis. This contraption will not specifically remove des-lys⁵⁸-β₂m but rather the whole β₂m- population from blood.

SUMMARY OF THE INVENTION

The present invention provides means for detecting and reducing the levels of des-Lys⁵⁸- β2-microglobulin in a complex environment on which both diagnostic and therapeutic applications may be based.

In a first aspect, the invention provides an antibody directed specifically against des-Lys⁵⁸- β2-microglobulin or a fragment of des-Lys⁵⁸-β2-microglobulin, the antibody being capable of forming a complex with des-Lys⁵⁸-β2-microglobulin also when des-lys⁵⁸-β₂m is present in a non-immobilised form. In preferred embodiments of this aspect, the antibody is a monoclonal IgG class antibody which is capable of forming a complex with des-Lys⁵⁸-β2- microglobulin present in solution and is suitable for use in a capture immunoassay. In further preferred embodiments, the antibody is generated by immunisation with one or more polypeptides comprising a sequence, which is located adjacent to the site of proteolytic cleavage of β₂microglobulin.

Another aspect of the invention provides a method for detection and/or quantification of des-Lys⁵⁸-β2-microglobulin present in a biological material, the method comprising the steps of contacting a sample of said biological material or an aliquot or fraction hereof with an antibody according to the invention and detecting any complexes of said antibody and des-Lys⁵⁸-β2-microglobulin formed. The method of the invention may be performed as an in vitro diagnostic method in particular for diagnosing or monitoring the development of an amyloid disease or for identifying subjects at risk or being predisposed for developing an amyloid disease. In preferred embodiments, the method is used for detection of des-Lys⁵⁸- β2-microglobulin in body tissues or fluids preferably of human origin. It is further preferred that the antibody:antigen complexes formed are detected by solid phase immunoassay, in particular a capture immnoassay such as an ELISA. In other preferred embodiments, the immune complexes are detected using immunoaffinity chromatography, preferably combined with mass spectrometry.

The term "subject" as used herein includes, but is not limited to, an organism; a mammal, including, e.g., a human, non-human primate (e.g., baboon, orangutan, monkey), mouse, pig, cow, goat, cat, rabbit, rat, guinea pig, hamster, horse, monkey, sheep, or other non- human mammal; a non-mammal, including, e.g., a non-mammalian vertebrate, such as a bird (e.g., a chicken or duck) or a fish, and a non-mammalian invertebrate.

While the method of the invention may be used to detect des-Lys⁵⁹-β2-microglobulin in relation to virtually any type of disease, it is preferred that the biological material subject to the method originates from a subject suffering from a disease or disorder selected from the group comprising kidney diseases, chronic inflammatory diseases, autoimmune diseases and cancer. In particular, use of the method is relevant in relation to hemodialysis.

Other aspects of the invention pertains to a pharmaceutical composition comprising an antibody as described above, and to such an antibody for use as a medicament as well as for the preparation of a medicament.

In addition, an aspect of the invention provides a method of preventing or reducing the formation of amyloid deposits, the method comprising a step of contacting a biological material with an antibody according to the invention in order to reduce the amount of free des-Lys⁵⁸-β2-microglobulin present in the biological material.

Still another aspect of the invention pertains to a method for generating an antibody according to the invention, the method comprising the steps of injecting a compound comprising an amino acid sequence of the invention into an animal under conditions allowing the generation of antibodies, isolating spleen cells from said animal and fusing the cells with a myeloma cell line, and screening the resulting hybridoma cell lines for specificreactivity with des-Lys^S8-β2-microglobulin.

Further, in an aspect of the invention, a device is provided for reducing the amount of des- Lys⁵⁸-β2-microglobulin present in a biological material, the device comprising an antibody according to the invention. Such a device may be an adjunct to a conventional dialyzer or it may be contemplated as an integrated part of such a device.

In a final aspect, the invention provides kits, such as in vitro diagnostic kits comprising the antibodies according to the invention.

DETAILED DISCLOSURE OF THE INVENTION

Amyloid deposition may be either a primary (idiopathic) process without known antecedent or secondary to some other condition and may be localized to one specific site or generalized throughout the body (systemic), usually with fatal consequences. Although considerable overlap is seen in the organ distribution of various forms of amyloid, primary amyloidosis tends to involve mesodermal tissues, most frequently affecting peripheral nerves, skin, tongue, joints, heart, and liver while secondary amyloidosis mainly affects parenchymatous organs, such as spleen, kidneys, liver, and adrenals. Amyloid deposits typically contain three components. Amyloid protein fibrils account for about 90% of the amyloid material and comprise one of several different types of proteins with the capacity to fold into what are called "beta-pleated" sheet fibrils, a unique protein configuration with binding sites for Congo red. In addition, amyloid deposits are intimately associated with the amyloid P (pentagonal) component (AP), a glycoprotein related to normal serum amyloid P (SAP), and are closely associated with sulfated glycosaminoglycans (GAG), complex carbohydrates of connective tissue.

Systemic forms of amyloid are derived from circulating protein precursors by conversion from soluble into insoluble (fibrillar) form. By convention, amyloid fibril types are designated by two letters: A for amyloid followed by a letter for the chemical type. There are two, chemically distinct, major types of amyloid protein fibrils designated AL and AA, respectively, and several minor types unrelated to AL or AA. AL (amyloid light chain) fibrils associated mainly with multiple myeloma are related to monoclonal immunoglobulin light chains synthesized by abnormal plasma cells. AA fibrils associated mainly with chronic inflammatory diseases are related to the nonimmunoglobulin amyloid associated (AA) protein and its serum precursor (SAA), an acute phase reactant synthesized by liver cells.

The classification of amyloidosis is based upon the tissue distribution of amyloid deposits (local or systemic amyloidosis), the absence or presence of preexisting disease (primary or secondary amyloidosis), and the chemical type of amyloid protein fibril.

Classifications of Amyloidosis:

Amyloid Clinical Associated Condition Fibril Precursor Classification Type

Systemic Amyloidosis Primary or Iglambda Multiple myeloma AL Secondary (or kappa chains) Chronic inflammatory disease Rheumatoid arthritis Secondary AA SAA Tuberculosis Skin and lung abscesses Cancer Secondary AA SAA Hodgkin's disease Secondary Hemodialysis for CRF(*) beta2-m beta2-m Primary Heredofamilial amyloidosis AA SAA Familial Mediterranean Fever Familial amyloid Transthy Transthyretin(#) polyneuropathy retin Localized Amyloidosis

Transthy Senile cardiac amyloidosis Transthyretin retin Amyloid Senile cerebral amyloidosis: Amyloid precursor beta Alzheimer's disease protein (APP) protein Endocrine tumors Procaltito Medullary carcinoma Calcitonin nin of thyroid Carpal tunnel syndrome, joint symptoms (DRA) deslysbeta-2-m/beta-2-m beta-2-m

(*): chronic renal failure; (#): transthyretin is a normal serum protein that transports thyroxin and retinol (vitamin A) and is deposited in a variant form.

AL type of amyloidosis occurs in about 5-10% of patients who have preexisting or coexisting multiple myeloma. Multiple myeloma is seen mainly in patients over 40 years of age (median age of 60 years) and, next to metastatic carcinoma, is the most common malignant tumor of bone. It is a malignant tumor of plasma cells which arises in the bone marrow, permeates the medullary cavity, erodes the bone cortex, and is characterized by multiple osteolytic lesions of vertebrae, skull, ribs, pelvis, and other bones and by narrow- banded electrophoretic peaks of monoclonal IgG (less commonly IgA , rarely IgD or IgE) in the serum and free light chains of the same kappa or lambda type in the urine (Bence- Jones proteinuria). An identical, patient-specific, free monoclonal light chain protein is also usually present in myeloma serum but, being smaller than albumin molecules, readily passes into the urine. Overall, about 70% of myeloma patients have both serum monoclonal Ig and urinary light chains, and the remaining patients have urinary light chains alone without serum monoclonal Ig.

The AL fibrils are derived from circulating light chains by proteolytic cleavage and conversion to an insoluble form. The organ distribution of AL deposits is usually generalized (systemic) and conforms to either the primary or secondary patterns previously noted. AL type of amyloidosis is also associated with some other rare monoclonal gammopathies (plasma cell/B immunocyte dyscrasias), such as solitary myeloma (of bone or soft tissue), Waldenstrom's macroglobulinemia, or heavy chain disease in which there are also sometimes an increased production of free light chains that become deposited as amyloid. Noteworthy, the majority of patients who develop AL type of amyloidosis apparently do so in the absence of clinically overt myeloma or other predisposing disease, and such cases are commonly referred to as primary or idiopathic amyloidosis. Nevertheless, in long term follow up, a substantial proportion of these patients do manifest overt, monoclonal Ig- producing plasma cell or lymphoid cell dyscrasias, such as myeloma, macroglobulinemia, or lymphoma.

Amyloidosis Associated with Inflammatory or Infectious Diseases: The amyloid deposits in this form of amyloidosis have a systemic distribution and contain AA (amyloid-associated) fibrils which are related to the nonimmunoglobulin AA protein and its serum protein precursor (SAA), an acute phase reactant synthesized by hepatic cells. Also called reactive or secondary amyloidosis, this form of amyloidosis occurs mainly as a complication of long standing inflammatory diseases, most frequently rheumatoid arthritis (A 5-10% of rheumatoid patients) and also dermatomyositis, scleroderma, regional enteritis, and ulcerative colitis.

Prior to the antibiotic era, chronic tissue-destructive infectious diseases, such as tuberculosis, chronic osteomyelitis, and bronchiectasis, were the most common antecedants of secondary amyloidosis. Now, amyloidosis often develops as a complication of skin and lung abscesses occurring in subcutaneous heroin abusers.

Reactive-type amyloidosis may also occur in association with cancer, such as Hodgkin's disease and renal cell carcinoma.

In addition, the following types of amyloids and disease associations are well known:

Amyloid associated with hemodialysis (AH): The systemic amyloid deposition of β2- microglobulin, a normal serum protein, occurs as a complication of long-term dialysis in patients with chronic renal failure because this protein does not pass through conventional dialysis membranes.

Amyloid associated with familial Mediterranean fever: The systemic deposition of AA fibrils occurs in familial Mediterranean fever, an autosomal recessive disorder seen in individuals of Sephardic Jewish, Armenian, and Arabic descent. Amyloid associated with familial amyloid neuropathies (AF): Amyloid deposition of a mutant form of transthyretin, a normal serum protein that transports thyroxin and retinol (vitamin A), occurs in peripheral nerves in familial amyloid polyneuropathy, an autosomal dominant disorder occurring in different parts of the world (Sweden, Portugal, Japan, and the U.S.).

Endocrine-related localized deposits of amyloid: Localized amyloid deposits are associated with hormones produced by certain endocrine tumors and endocrine glands, such as medullary carcinoma of the thyroid gland (procalcitonin), islet cell tumors of the pancreas, and the islets of Langerhans (islet associated polypeptide, IAPP) in patients with type II diabetes mellitus.

Age-related localized deposits of amyloid: Amyloid deposits of transthyretin occur in the heart of elderly patients with senile cardiac amyloidosis. Beta amyloid protein is deposited in the cerebral blood vessels and plaques of patients with senile cerebral amyloidosis and Alzheimer's disease.

The present invention is based on the previous observation that, in chronic renal disease and in particular in long-term hemodialysis treatment, a fraction of the circulating β2- microglobulin is modified from the single-chain 99-amino acid polypeptide into a heterodimeric disulfide-linked 98 amino acid protein, des-Lys⁵⁸-β2-microglobulin, which comprises the two amino acid sequences

H-Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-Asn-Gly-Lys-Ser-Asn- Phe-Leu-Asn-Cys-Tyr-Val-Ser-Gly-Phe-His-Pro-Ser-Asp-Ile-Glu-Val-Asp-Leu-Leu-Lys-Asn- Gly-Glu-Arg-Ile-Glu-Lys-Val-Glu-His-Ser-Asp-Leu-Ser-Phe-Ser-OH,

and

H-Asp-Trp-Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr-Thr-Glu-Phe-Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr- Ala-Cys-Arg-Val-Asn-His-Val-Thr-Leu-Ser-Gln-Pro-Lys-Ile-Val-Lys-Trp-Asp-Arg-Asp-Met- OH

connected by a single disulfide bond.

These amino acid sequences are provided as SEQ ID NO. : 1 and 2, respectively, and are presented below in one-letter code: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFS

DWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

The invention further relates to the fact that, in spite of the correlation between the levels of des-Lys⁵⁸-β2-microglobulin and the generation of amyloid deposits during long-term hemodialysis treatment, practical means for determining the levels of circulating des-Lys⁵⁸- β2-microglobulin and lowering these levels have not yet been developed.

In the broadest sense, the invention provides reliable, rapid and technically simple means for detecting and reducing the levels of des-Lys⁵⁸-β2-microglobulin in a complex environment. Such means are required for diagnosis, screening, monitoring and treatment of the formation of amyloid deposits. It is easy to contemplate techniques for capturing an antigen or a target molecule in a complex environment by the use of antibodies, however, the requirements for such antibodies are not easily met.

While the antibodies of the present invention must be specific i.e. only binding to the target molecule in the presence of high amounts of many unrelated molecules, they must also be able to recognise the antigen or the target molecule in one of its native forms. In particular, the antibodies should not require the conformation of the antigen be stabilised or unfolded by immobilisation.

Accordingly, one main aspect the present invention provides an antibody directed specifically against the target molecule des-Lys⁵⁸-β2-microglobulin or a fragment of des- Lys⁵⁸-β2-microglobulin, said antibody being capable of forming a complex with des-Lys⁵⁸- β2-microglobulin present in a non-immobilised as well as in an immobilised form.

In the present context, 'immobilisation' or 'immobilised' refers to the direct association of an antigen or target molecule support materials by either covalent or non-covalent bonds. Generally, molecules may be immobilised either passively through hydrophobic or ionic interactions or covalently by attachment to activated surface groups. 'Immobilisation' does not comprise the capture of the antigen or target molecule by antibodies present on the support.

The immobilisation of an antigen or target molecule may lead to structural changes such as unfolding of the molecule. In a particular embodiment of the invention, the antibody of the present invention is thus functionally characterised and selected so that it is also capable of forming a complex with des-Lys⁵⁸-β2-microglobulin being in solution and so having a conformation, which has not been reached by unfolding of the polypeptide in its native conformation e.g. by immobilisation on a surface.

In most complex environments, such as the environment wherein it is naturally present, the antigen or target molecule will be present in a form of solution. Furthermore, upon isolation from its native environment the target molecule may be present in a solution, for instance a buffered solution such as a tissue extract, or a fraction of a body fluid or simply a solution of salts. It may also be desirable to capture the antigen during a purification procedure such as a chromatography procedure. Therefore, in a preferred embodiment of the present invention, the antibody is capable of forming a complex with des-Lys⁵⁸-β2- microglobulin present in solution.

The antibody of the present invention is specific for des-Lys⁵⁸-β2-microglobulin in that it does not recognise and form complexes with β2-microglobulin. The antibody may therefore be directed against epitopes present on any of the two amino acid sequences formed upon cleavage of β2-microglobulin and removal of Lys58 as well as any part of these sequences. In a preferred embodiment, the antibody according to the invention is obtained by immunising an animal with one or more polypeptides comprising the sequence of SEQ ID NO. : 1 and/or SEQ ID NO. : 2 or a part hereof.

As used herein, "epitope" refers to an antigenic determinant of a polypeptide. An epitope could comprise 3 amino acids in a spatial conformation, which is unique to the epitope. Generally an epitope consists of at least 5 such amino acids, and more usually, consists of at least 8-10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and 2- dimensional nuclear magnetic resonance.

The antibody of the invention may be a polyclonal antibody in which case it is obtained from an animal which has been immunised with one or more polypeptides comprising the sequence of SEQ ID NO. : 1 and/or SEQ ID NO. : 2 or a part hereof. In the present context the term 'polyclonal antibody' refers to a serum containing mixed populations of antibodies of various affinities binding to one or several epitopes in an antigen.

The term "polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

When seeking to generate antibodies which will recognise a given target molecule in its native conformation a preferred strategy is to direct the antibody against a part of the target molecule that is exposed on its surface or protrudes from its surface. Based on the structure of des-Lys⁵⁸-β2-microglobulin as presented above, the N- and C-terminal ends of the two amino acid sequences of which the polypeptide is composed and which are not present in normal β2-microglobulin appear as possible target regions for an antibody. Therefore, the antibody according to the invention is preferably obtained from an animal, which has been immunised with one or more polypeptides comprising a C-terminal and/or an N-terminal part of said sequences. In order to ensure specificity of the antibodies towards des-Lys⁵⁸-β2-microglobulin and minimise cross-reactivity with β2-microglobulin, it is preferred that the antibodies are directed towards neo-epitopes, which are revealed upon proteolytic cleavage of β2-microglobulin. In particular, amino acid sequences corresponding to the sequences of β2-microglobulin, which are adjacent to the site of proteolytic cleavage, will be useful as antigens when generating the antibodies according to the invention.

In the present context it is further preferred that the antibody is obtained from an animal, which has been immunised with one or more polypeptides comprising the sequence of SEQ ID NO. : 1 or a part hereof. Especially, it is preferred that the antibody is obtained from an animal which has been immunised with an amino acid sequence which corresponds to a C- terminal part of SEQ ID NO. : 1. Example 1 of the present application describes a procedure for the generation of an antibody according to the invention. In this procedure, a construct comprising a synthetic peptide corresponding to the 9 C-terminal amino acid residues of SEQ ID NO. : 1 is used as antigen. A sequence corresponding to these 9 amino acids is provided as SEQ ID NO. : 3 and, currently, an antibody obtained from an animal immunised with an amino acid sequence corresponding to SEQ ID NO. : 3 is the most preferred embodiment of the invention.

It is apparent that the polypeptides or amino acid sequences which are used for immunisation may comprise a fragment or fragments of SEQ ID NO. : l less than or equal to any length between 6 and 57 amino acids, such as but not limited to less than or equal to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 amino acids. It further appears that these fragments of SEQ ID NO. : l may have a length of 6 - 8, 6 - 10, 6 - 12, 6 - 15, 6 - 17, 6 - 20, 6 - 22, 6 - 25, 6 - 27, 6 - 30, 6 - 32, 6 - 35, 6 - 37, 6 - 40, 6 - 42, 6 - 45, 6 - 47, 6 - 50, 6 - 52, 6 - 55 or 6 - 57 amino acids.

For all the aspects of the invention, it is thus apparent that the polypeptides or amino acid sequences of the invention may include sequences, which show anywhere between 1- 100% sequence identity, such as at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or preferably 100% sequence identity to SEQ ID NO. : 1 or SEQ ID NO. : 2 or a fragment or sub-sequence thereof.

The term 'sequence identity' indicates a quantitative measure of the degree of homology between two amino acid sequences or between two nucleic acid sequences of equal length. If the two sequences to be compared are not of equal length they must be aligned to give the best possible fit, allowing the insertion of gaps or, alternatively, truncation at the ends of the polypeptide sequences or nucleotide sequences. The sequence identity can be calculated as ., — , wherein N_dlf is the total number of non-identical residues in the two sequences when aligned and wherein N_ref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (N_dlf=2 and N_ref=8). A gap is counted as non-identity of the specific residue(s), i.e. the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (N_dlf=2 and N_ref=8).

In all embodiments of the invention the percentage of sequence identity between one or more sequences is based on alignment of the respective sequences as performed by clustalW software (http:/www. ebi.ac.uk/clustalW/index. html) using the default settings of the program. These settings are as follows: Alignment=3Dfull, Gap Open 10.00, Gap Ext. 0.20, Gap separation Dist. 4, Protein weight matrix: Gonnet.

Amino acid sequences corresponding to part or parts of des-Lys⁵⁸-β2-microglobulin can be obtained by the use of conventional techniques for generating recombinant polypeptides. As an alternative, such amino acid sequences may also be provided by use of the well- known methods of solid or liquid phase peptide synthesis.

For immunisation with a relatively short polypeptide which, in itself, may not be sufficiently immunogenic to elicit an antibody response, it may be preferred to use chimeric molecules comprising the polypeptide fused to a powerful immunogenic molecule such as tuberculin or keyhole limpet hemocyanin resulting in the production of antibodies against this chimeric molecule. Optionally, multiple repeats of the antigenic polypeptide may be present in the chimeric molecule. Examples of immunogenic molecules which may be used according to the invention comprise keyhole limpet hemocyanin and ovalbumin. In a preferred embodiment, the immunogenic molecule is tuberculin. Alternatively, short peptides for immunisation may be part of a composition further comprising one or more adjuvants, for example, aluminim hodroxide, aluminium phosphate, MPL™, QS-21 (Stimulon™) or incomplete or complete Freund's ajuvant.

The antibodies of the invention may include, but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. A person skilled in the art knows that antibodies can be produced by immunisation of various hosts including goats, rabbits, rats, and mice.

When generating polyclonal antibodies, serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an antigen of interest also contains antibodies to other antigens, the polyclonal antibodies can be isolated and purified by immunoaffinity chromatography.

The term "serum" is used in its normal meaning, i.e. as blood plasma without fibrinogen and other clotting factors.

In the context of the present invention the term 'isolated' requires the material to be removed from the environment in which it was present originally. For example, a polypeptide or nucleic acid, which is expressed in a cell, is not isolated. However, the same polypeptide or nucleic acid, when separated from some or all of the coexisting material occurring in the original environment, will be considered as isolated. It is in accordance with this definition to regard polypeptides and antibodies present in cell lysates as isolated. By 'purifying' a compound such as an antibody is meant increasing the degree of purity of a preparation of the compound by removing completely or partially at least one contaminant from the preparation. When applied to a preparation of a compound the term 'degree of purity' refers to its relative content by weight of the compound of interest, based on the total weight of the preparation. The degree of purity of a compound may be within the range of 1 - 100%, such as from 1 - 100%, 10 - 100%, 20 - 100%, 30 - 100%, 40 - 100%, 50 - 100%, 60 - 100%, 70 - 100%, 80 - 100% and 90 - 100%. 'Substantially pure' is herein used to describe a polypeptide or a nucleic acid with a degree of purity of at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 99% or preferably substantially pure from other components. The % value herein indicates %(w/w). It is particularly preferred that the antibody of the invention is a monoclonal antibody. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and variations in post translational modifications that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies can be produced using any of the conventional techniques that provide for the production of antibodies from cell lines in continuous culture. The first step in a conventional approach for production of murine monoclonal antibodies is to immunize a mouse with an antigen. When the mouse begins to produce antibodies to the antigen, its spleen is removed. Antibody-producing cells from the spleen are then fused with a myeloma cell line, one which is not antibody-producing and has been maintained in culture. The new fused cell line, which does produce antibodies, is grown in culture. Finally, the hybridoma fluid, which contains monoclonal antibodies, is harvested from the cell culture. Specialised techniques for production of monoclonal antibodies include the human B-cell hybridoma technique, and the EBV-hybridoma technique.

In the present specification and claims, the term antibody is used in its normal meaning referring to glycoproteins, which exhibit binding specificity to a specific antigen. Antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V_H) at one end followed by a number of constant domains. Each light chain has a variable domain at one end (V_L) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.

The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. The variability is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each with a single antigen binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')₂ fragment that has two antigen combining sites and is still capable of cross-linking antigen. "Fv" is the minimum antibody fragment, which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V_H-V_L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of a Fv comprising only three CDRs specific for an antigen) i.e. an scFv, has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments which have disulfide bridges between them.

The "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes of which there are five major classes: IgA, IgD, IgE, IgG and IgM. Several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. For use in quantitative immunoassays high affinity monoclonal antibodies (K_a > 10⁷ M^"1), usually of IgG class, with validated specificity in relevant matrices (e.g., plasma and serum) are required. A consequence of these requirements is that IgM class monoclonal antibodies are not optimal and will typically be avoided. This is because IgM antibodies - even if they display the desired specificity - most often are of lower affinity than IgG antibodies. This is due to the fact that IgM antibodies are part of the primary immune response to antigens and participate little in the secondary responses where Ig class switching takes place, i.e. they display less somatic hypermutation and secondary clonal selection. Also, the five times larger size of IgM antibodies make them less diffusible and simply for sterical reasons make them less suited for the multilayer (e.g. capture ELISA) techniques necessary for quantitatively analyzing biomolecules in complex environments such as serum. Finally, the large size of IgM also in some cases precludes access to epitopes buried in natively folded protein structures, i.e, reactivity will depend on some degree of unfolding of the target molecule.

Therefor in a further preferred embodiment of the present invention the antibody is an IgG class antibody. In an even more preferred embodiment the antibody is an IgG-1 antibody possessing -type light chains. A currently preferred embodiment of the invention is the antibody produced by any of the hybridoma cell lines denoted 4-G3, 10-B5, and 13-D5. The antibodies are identified as 332-01, 332-02, 332-03. In a most preferred embodiment, the antibody is identified as 332-01.

The term "antibody and antibody fragments" is used in the present specification and claims in the broadest sense and specifically covers single monoclonal antibodies as well as anti- body fragments (e.g., Fab, Fab', F(ab')₂, Fv and scFv), as long as they specifically recognize the antigen. The invention thus relates to methods according to the invention wherein the antibody fragment is selected from the group consisting of Fab, Fab ^' , F(ab ')₂, Fv, scFv and other antigen-binding sub-sequences of an antibody. Complexes of the antibodies or fragments of antibodies of the invention are also within the scope of the inven- tion.

Alternatively, antibodies, such as chimeric antibodies, and any antibody fragments corresponding to antibodies generated in response to immunisation with the amino acid sequences of the invention or parts of such antibodies can be produced by recombinant processes well known in the art. Preferred antibody fragments do not contain the Fc region of the antibody molecule. The Fc region is responsible for effector functions of the immunoglobulin (Ig) molecule, such as complement fixation, allergic responses and killer T cell activation. The smaller size of the antibody fragment may help improve tissue bioavailability, which may be critical for better dose accumulation in disease indications. Furthermore, they have reduced immunogenicity, they do not induce precipitation (Fab only) and they can be used for a variety of in vivo applications and immunoassays.

Antibody fragments can be produced via recombinant methods creating single chain antibodies ("ScFv"), in which the heavy and light chain Fv regions are connected, or by enzymatic digestion of whole antibody. In particular, Fabs can be converted to whole Ig molecules. The light-chain gene and variable gene fragment of the heavy-chain sequence of each clone can be inserted into a eukaryotic expression vector containing a Ig constant region gene, for instance of human origin. Such chimeric antibodies, which are of partially human origin are less immunogenic than wholly murine MAbs, and the fragments and single chain antibodies are also less immunogenic. All these types of antibodies are therefore less likely to evoke an immune or allergic response. Consequently, they are better suited for in vivo administration in humans than wholly animal antibodies, especially when repeated or long-term administration is necessary.

Humanized antibodies have a greater degree of human peptide sequences than do chimeric antibodies. In a humanized antibody, only the complementarity determining regions (CDRs) which are responsible for antigen binding and specificity are animal derived and have an amino acid sequence corresponding to the animal antibody, and substantially all of the remaining portions of the molecule (except, in some cases, small portions of the framework regions within the variable region) are human derived and correspond in amino acid sequence to a human antibody. In addition to chimeric antibodies, such humanised antibodies may be preferred for any therapeutic applications according to the present invention.

Finally, antibodies according to the invention may also be prepared as human antibodies using a number of methods known in the art, as for instance the trioma methodology. According to this approach, antibodies are produced in so-called triomas, cells that are generated by fusing a mouse myelome cell line with a human B-lymphocyte to form a xenogeneic hybrid cell, which then is fused with an immunised human B-lymphocyte. Alternatively, human antibodies may be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobolin locus, which preferably, includes unrearranged sequences of heavy and light chain components. Inactivation of endogenous immunoglobulin genes and introduction of exogenous immunoglobulin genes can be achieved by targeted homologous recombination, or by introduction of yeast artificial chromosomes. The transgenic animals resulting from this process are capable of functionally rearranging the immunoglobulin component sequences expressing a repertoire of antibodies of various isotypes. Finally, human antibodies can be obtained by phage display screening methods involving the screening of a DNA library from human B-cells and other in vitro selection techniques (e.g. ribodisplay).

Due to their ability to form complexes with the antigen in non-immobilised form the antibodies of the present invention are suitable for use in a capture immunoassay. In a capture or sandwich immunoassay, a polyclonal antibody is used as a capturing agent collecting or capturing target molecules from a complex environment. In the present context the polyclonal antibody may be an antibody directed against β2-microglobulin which is unable distinguish between the various β2-microglobulin species allowing the isolation of the entire β2-microglobulin family from complicated sample matrices. Subsequent to this initial capturing step the captured des-Lys⁵⁸-β2-microglobulin can be detected using the specific antibodies according to the invention with no substantial interference from other protein or peptide components of the environment. In further assay steps, the complexes formed between antibodies of the invention and the captured antigen are detected by the use of conventional secondary immunoreagents including labelled secondary antibodies or antibody conjugates directed against the des-Lys⁵⁸-β2- microglobulin specific antibodies according to the invention.

In a capture immunoassay, the capturing step has a concentrating effect enabling the detection of relative low amounts of target molecule in a complex environment. Since des- Lys⁵⁸-β2-microglobulin is normally present in low concentrations as compared to β2- microglobulin, this concentrating effect greatly improves the practicability of detecting des- Lys⁵⁸-β2-microglobulin in samples of biological origin.

In addition to being usable in capture immunoassays, the antibodies of the present invention may be used in virtually all types of immunosorbent assays and solution assays such as affinity electrophoresis, affinity chromatography, other assay types such as surface plasmon resonance interaction measurements, equilibrium dialysis, fluorescence quenching/enhancement spectroscopy, isothermal calorimetry, immunosensors, indirect immunofluorescence, radioimmunoprecipitation assays, in vivo immunoscintigraphy and immunotargeting.

As an alternative to the use of secondary reporter antibodies or antibody conjugates, the antibodies according to the invention may themselves be provided with a detectable label or reporter group. Whether present in the specific des-Lys-β2-microglobulin-directed antibodies of the invention or on secondary reporter antibodies, reporter groups comprise enzymes, radioactive isotopes and non-radioactive compounds such as fluorescent or chemiluminescent compounds. Detection methods comprise the use of fluorescent or chemiluminescent compounds due to the high sensitivity and low background of such methods. Non-limiting examples of fluorescent compounds, which may be used for the purposes of the present invention are FITC, phycoerythrin(PE), Texas red, a Cy3 or a Cy5 conjugate, green fluorescence protein (GFP) or modifications thereof. Examples of suitable enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). The amount of signal produced by such reporter groups can be detected using conventional techniques and apparatus, which detects e.g. the amount of light emitted at a certain wavelength. Conventional apparatus includes ELISA readers, spectrophotometers, absorbance reader, photomultiplier, Charge Coupled Device (CCD), combined with a source of illumination, such as a bulb, light emitting diode (LED), laser and optional filters. In a currently most preferred aspect of the invention, detection of the complexes of des- Lys-β2-microglobulin-directed antibodies and des-Lys⁵⁸-β2-microglobulin are detected using alkaline phosphatase labelled goat anti-mouse immunoglobulin antibodies and the alkaline phosphatase activity is visualised using p-nitrophenyl phosphate as the enzyme substrate.

As mentioned, the reporter groups may also be labelled with radioactive isotopes such as ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³³P, or ³²P and subsequently detected by radioactivity counters or autoradiography. This method is not the most preferred due to the inherent dangers associated with the use of radioactive isotops.

Another main aspect of the present invention pertains to a method for detection and/or quantification of des-Lys⁵⁸-β2-microglobulin present in a biological material including tissue samples, the method comprising the steps of contacting a sample of said biological material or an aliquot or fraction hereof with an antibody as described in any of the embodiments above and detecting any complexes of said antibody and des-Lys⁵⁸-β2- microglobulin formed. This method may be used simply to detect any des-Lys⁵⁸-β2- microglobulin present in a biological material and to determine its concentration. However, in a preferred embodiment of the invention, the method is incorporated as part of an in vitro diagnostic procedure in which the determined concentrations of des-Lys⁵⁸-β2- microglobulin are correlated to concentrations of des-Lys⁵⁸-β2-microglobulin present in a body tissue or fluid and where the levels of des-Lys⁵⁸-β2-microglobulin, when compared to known reference values if desired, may be indicative of a subject being pre-disposed to amyloidosis or suffering from amyloidosis. Such a diagnostic procedure may also be used for monitoring the levels of des-Lys⁵⁸-β2-microglobulin during treating a patient suffering from amyloisidosis. It is contemplated that monitoring the levels of des-Lys⁵⁸-β2- microglobulin during treatment of amyloid diseases will greatly improve the quality of such treatment. The embodiments of a method for detecting and/or quantifying des-Lys⁵⁸-β2- microglobulin as well as the characterising features described in the following may thus apply by analogy to an in vitro procedure for diagnosing amyloisidosis.

In the present context, the term 'diagnosing' and 'diagnostic' includes determining whether a person has an amyloid disease or is at risk or being pre-disposed for developing an amyloid disease as well as indicating the stage or prognosis of an amyloid disease in a patient.

In addition to being used in assays for detecting their target molecule in solution the antibodies according to the present invention may also be used directly on a tissue sample. By 'tissue sample' is meant histological specimens such as biopsies, resections, and other material from the body or from a body tissue. Accordingly, one aspect of the invention pertains to an in vitro diagnostic method, wherein said biological material is a body tissue. For the purposes of the present invention it is of particular relevance to detect des-Lys⁵⁸- β2-microglobulin in samples of amyloid body tissues such as in fat biopsies, connective tisue specimens and in kidney-, liver-, and muscle biopsies. The detection of complexes of des-Lys⁵⁸-β2-microglobulin and the antibodies according to the invention may be accomplished using standard immunohistochemical techniques known in the art.

As described above, the antibodies according to the present invention are also useful in assays for detection of des-Lys⁵⁸-β2-microglobulin in solution and may therefore readily be applied to the detection of des-Lys⁵⁸-β2-microglobulin present in body fluids. Accordingly, a method for detection of des-Lys⁵⁸-β2-microglobulin a body fluid is a preferred embodiment of the invention.

Two main classes of body fluids exist; intracellular and the extracellular fluid, the latter comprising interstitial fluid, plasma and transcellular fluid such as digestive juice and mucus and it readily appears that the method of the invention can be used for detecting and quantifying the content of des-Lys⁵⁸-β2-microglobulin in any if these fluids. A further preferred embodiment of the invention pertains to a method for detection and/or quantification of des-Lys⁵⁸-β2-microglobulin present in a biological material, wherein the biological material is a body fluid selected from the group comprising blood, serum, saliva, urine, semen, spinal fluid, joint fluid, pleural exudate, ascites, dialysis fluid and lymphatic fluid. It may be even further preferred that the biological material is blood or serum.

While it is contemplated that the antibodies of the present invention may be used for detection of des-Lys⁵⁸-β2-microglobulin in samples derived from virtually any mammalian organism it readily appears that the primary field of application relates to the detection of des-Lys⁵⁸-β2-microglobulin in humans. Therefore, a method according to the invention, wherein the biological material is of human origin constitutes another preferred embodiment of the invention.

As discussed above, des-Lys⁵⁸-β2-microglobulin is generally present in relatively limited amounts. Preferred embodiments of the invention therefore relate to procedures for detecting des-Lys⁵⁸-β2-microglobulin in a fraction of a biological material in which the des- Lys⁵⁸-β2-microglobulin concentration is already increased relative to the amounts present in the biological material. Such fractions can be generated in many ways, for instance by separating constituents of the biological material according to size. However, the concentrating procedure is most conveniently performed in a capture step using antibodies capable of binding to β2-microglobulin species.

Another preferred embodiment of the invention is a method as described above wherein the complex formed by des-Lys⁵⁸-β2-microglobulin and the antibody according to the invention is detected in a solid phase immunoassay. There are many carriers to which antibodies or antigens can be coupled. Non limiting examples include glass, polystyrene, polyethylene, polypropylene, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses and magnetite. For the purposes of the present invention, the preferred solid phase is a commercially available polystyrene microtiter plate. It is further preferred that the solid phase is coated with an antibody capable of binding β2- microglobulin species, including des-Lys⁵⁸-β2-microglobulin.

Briefly, there are 3 types of immunoassays: Enzyme immunoassay (EIA; ELISA), Radioimmunoassay (RIA) and Fluorescent immunoassay (FIA) is used most often for environmental field analysis because it can be optimized for speed, sensitivity, and selectivity, and because it contains no radioactive materials.

The enzyme immunoassay is based on the use of enzyme-conjugated antibodies or antigen for the detection of antigen :antibody reactions. A variant of the assay relies on competition between unconjugated antigen and enzyme conjugated antigen for binding to a limited amount of antiserum or antibody sites.

The basic principle of a radioimmunoassay (RIA) is the use of radiolabelled antibodies or antigens to detect antigen:antibody reactions. The antibodies or antigens are labelled with the a radioactive isotope, and the presence of antigen :antibody reactions is detected using a gamma counter. RIAs can be performed in solution as well on filters. In solution the antigen:antibody complexes are precipitate and the amount of radioactivity in the supernatant is measured.

Enzyme-linked-immunosorbent assay relies on an enzymatic conversion reaction and is effectively used to detect the presence of specific substances. One type of ELISA is the two-antibody "sandwich" ELISA. This assay is used to determine the antigen concentration in samples. The assay is performed by coating a microtiter plate with antibody, antigen is then added and allowed to complex with the bound antibody. Unbound products are subsequently removed with a wash, and a labelled second antibody (the "detection" antibody) is allowed to bind to the antigen, thus completing the "sandwich". The assay is then quantitated by measuring the amount of labelled second antibody bound to the matrix, through the use of a colorimetric substrate. Another embodiment of the said technology concerns the use of an anti-desLys58-β2m antibody to capture the antigen from a solution followed by probing with patient samples for patient antibodies specific for the antigen.

A variant of the solid phase immunoassay, which may applied to diagnostic procedures is the so-called dip-stick test. It is a method of detecting specific antigen or antibody from a bodily fluid sample. A nucleic acid, antigen or antibody is bound to the membrane of the dip-stick and contact to a labelled or unlabelled bodily fluid is allowed for a given time. The nucleic acid, antigen or antibody bound on the membrane can in some methods be hybridised to antigen or antibody labelled with a dye.

As suggested above the complexes of antibody and des-Lys⁵⁸-β2-microglobulin may be detected in a capture immunoassay, an approach, which is currently a preferred embodiment of the invention. It is even more preferred that the capture immunoassay is in the form of an ELISA.

As an alternative to use of the antibody according to the invention in a solid phase immunoassay as described above the antibody may also be used for instance for assay types wherein complexes of the antigens and antibodies are formed in solution, such as in applications relying on immunoprecipitation. The antibody according to the invention may also be useful for immunoaffinity chromatography due to its ability to bind the antigen in solution. In a specific embodimentof the invention, therefore, the complex formed between the antibody and des-Lys⁵⁸-β2-microglobulin is detected by immunoaffinity chromatography.

Another preferred embodiment of the invention pertains to a method as described above and in which the complex of antibody and des-Lys⁵⁸-β2-microglobulin or des-Lys⁵⁸-β2- microglobulin alone is detected by mass spectrometry. The theory of mass spectrometry is well known in the art but, briefly, mass spectrometry uses the difference in mass-to- charge ratio (m/z) of ionized atoms or molecules to separate them from each other. Mass spectrometry is therefore useful for quantitation molecules and also for determining chemical and structural information about molecules. Molecules have distinctive fragmentation patterns that provide structural information.

In general, a mass spectrometer consists of an ion source, a mass-selective analyzer, and an ion detector. Since mass spectrometers create and manipulate gas-phase ions, they operate in a high-vacuum system. Five conventional mass analyzer designs are available: Fourier-transform mass, ion-trap, magnetic sector, quadrupole and time-of-flight mass spectrometers. The magnetic-sector, quadrupole, and time-of-flight designs also require extraction and acceleration ion optics to transfer ions from the source region into the mass analyzer.

It is contemplated that any of the types of mass spectrometry mentioned above will be able to provide sensitive and reliable detection of complexes of des-Lys⁵⁸-β2-microglobulin and antibody according to the invention or detection of des-Lys⁵⁸-β2-microglobulin that has been isolated or purified using the antibody of the invention. However, a preferred approach for detecting des-Lys⁵⁸-β2-microglobulin or des-Lys⁵⁸-β2-microglobulin:antibody complexes is the use of time-of-flight mass spectrometry.

Since direct analysis of non-purified or crudely separated material by mass spectrometry is possible for small compounds it is not well suited for complex biological materials. It is therefore desired to combine detection by mass spectrometry with purification means based on liquid chromatography, preferably immunoaffinity chromatography. For the sake of convenience, the steps of isolation and/or purification and detection of the des-Lys⁵⁸-β2- microglobulin may be arranged as an on-line coupling of the immunochromatography equipment and the mass spectrometer. Currently, a preferred se-up is an immunoaffinity- liquid chromatgraphy-electrospray ionization mass spectrometry system as described by Sen et al., 2000.

According to the conventional classification of amyloid presented above the presence of des-Lys⁵⁸-β2-microglobulin is mainly connected to dialysis-related amyloidosis. In addition, des-Lys⁵⁸-β2-microglobulin has also been detected in connection with a variety of cancers and disorders of the immune system. The involvement of des-Lys⁵⁸-β2-microglobulin in these types of diseases is far from fully understood. However, the correlation between the presence of des-Lys⁵⁸-β2-microglobulin and the formation of amyloid deposits clearly merits the application of the antibody and the method according to the invention for detection of antibodies in material derived from subjects suffering from virtually all disease classes including congenital and hereditary diseases, allergies and inflammatory diseases, degenerative or chronic diseases, metabolic diseases and cancer/neoplastic diseases. A preferred embodiment of the invention, though, is a method according to the invention wherein said biological material originates from a subject suffering from a disease or disorder selected from the group comprising kidney diseases, chronic inflammatory diseases, autoimmune diseases and cancer.

A primary objective of the present invention, however, is to provide means for improving the treatment of any disease requiring hemodialysis, and it is thus preferred that the biological material used in the method according to the invention originates from a subject undergoing hemodialysis. As the formation of amyloid deposits is a particular concern in relation to diseases requiring long-term hemodialysis, such as chronic renal failure, the method according to the invention may logically be incorporated into treatment plans for such diseases.

As mentioned above the method of the invention may be an in vitro diagnostic method for diagnosing an amyloid disease or susceptibility to an amyloid disease. The amyloid disease may be selected from the group comprising any of the above-mentioned primary and secondary amyloid diseases. In particular, the in vitro diagnostic method may be used for diagnosing amyloidosis associated with hemodialysis.

An aspect of the present invention relates to therapeutic antibodies. Such antibodies may form parts of compositions which can be administrated to a subject, preferably a subject suffering from an amyloid disease, a subject being at risk of developing an amyloid disease or a subject likely to develop complications from chronic disease, where such complications comprise the generation of amyloid deposits.

In a pharmaceutical composition an effective amount of the antibody according to the invention may be present in combination with pharmaceutically acceptable additives. For intravenous administration the preferred carriers are physiological saline or phosphate buffered saline. For parenteral, subcutaneous, intradermal or topical administration the formulation may include a sterile diluent, buffers, regulators of tonicity and antibacterials. The active compound may be prepared with carriers that protect against degradation or immediate elimination from the body, including implants or microcapsules with controlled release properties. It is further understood that a pharmaceutical composition must be clinically safe and free of any putative infectious agents such as virus and bacteria. Elimination of virus and bacteria can be achieved via filtration or addition of anti-virusidal and anti-bacterial agents known in the art.

Another aspect of the invention pertains to a method of preventing or reducing the formation of amyloid deposits, the method comprising a step of contacting a biological material with an antibody according to the invention in order to reduce the amount of soluble des-Lys⁵⁸-β2-microglobulin present in said biological material. It is to be understood that the method may comprise administrating a pharmaceutical composition as described above to a subject that has developed amyloid deposits or is at risk or pre- disposed for developing amyloid deposits. Dosis regimens and administration schemes will be determined by the nature and severity of the disease or condition to be treated. Still other aspects of the invention pertain to an antibody as described above for use as a medicament and to the use of an antibody according to the invention for the preparation of a medicament.

Furthermore the invention also provides a method for generating an antibody according to the invention. Most notably this method leads to the generation of antibodies which are specific for des-Lys⁵⁸-β2-microglobulin and which do not cross-react with β2-microglobulin.

More specifically, the method comprises the step of injecting one or more of the polypeptides described above into an animal as part of a procedure for generating polyclonal or monoclonal antibodies as well as fragments of such antibodies. Preferably, the procedure leads to the generation of monoclonal antibodies.

In a preferred embodiment, the method comprises the steps of a) injecting into an animal a compound comprising a peptide of the sequence Cys- Val-Glu-His-Ser-Asp-Leu-Ser-Phe-Ser (SEQ ID NO. :3) under conditions allowing the generation of antibodies, b) isolating spleen cells from said animal and fusing the cells with a myeloma cell line, c) screening the resulting hybridoma cell lines for reactivity with des-Lys⁵⁸-β2- microglobulin.

It is further preferred to generate the antibodies in mice, most preferably NMRI female mice.

In other aspects of, the present invention provides a device for reducing the amount of des-Lys⁵⁸-β2-microglobulin present in a biological material, the device comprising an antibody according to any of the embodiments of the invention described above. In general terms, such a device may comprise means for contacting the antibody with the biological material. Such means may be any form of container, reservoir, tubing, hosing, measuring devise and dispenser. Furthermore, the device may comprise means for detaining the antibody or any complex of antigen and antibody formed from the biological material. Means for detaining the antibody may for instance be semi-solid or solid phase or supports such as those described above in relation to solid phase immunoassays, or the means may be filters, membranes, or gel materials. It is contemplated that the antibody according to the invention, when located on such means, will form an adsorbent capable of removing or detaining antibody-bound des-Lys⁵⁸-β2-microglobulin from a biological material. Accordingly, in a preferred embodiment, the device comprises an antibody which is attached to a solid support. In further preferred embodiments the device has the form of an adjunctive hemoperfusion device. Such a hemoperfusion device may be constructed as an independent unit or it may be an integrated part of conventional dialyzer. The hemoperfusion device may thus be designed specifically to remove des-Lys⁵⁸-β2-microglobulin from the blood in hemodialysis patients.

Dialyzers constructed with cuprophane dialysis membranes (cellulosic), long the standard therapy for chronic dialysis, are incapable of reducing the concentrations of β₂- microglobulin and des-Lys⁵⁸-β2-microglobulin and may in fact alter the conformational structure of β₂-microglobulin, promoting amyloidosis. On the other hand non-cellulosic dialyzer membranes, e.g. polyacrylonitrile, and polysulfone membranes, may reduce β₂- microglobulin concentrations during treatment, by a combination of adsorption (the majority) to the membrane and by convective removal (the minority), the latter of which is dependent on high rates of ultrafiltration. The efficiency of the latter membranes is not considered optimal, and usually adsorption is complete early in the dialysis procedure. Moreover, dialyzer reuse significantly impairs the removal of β₂-microglobulin.

Hemoperfusion devices containing adsorbents, have been used to enhance the removal of 'middle molecule' amino acids of which β2-microglobulin is a representative, and creatinine, in dialysis patients, using non-specific agents such as activated charcoal. A porous resin hemoperfusion device has also been shown to reduce β₂-microglobulin concentrations during extracorporeal treatment, with improvement in the clinical manifestations of dialysis related amyloidosis. The sorbent comprising specific des-Lys⁵⁸- β2-microglobulin antibodies according to the invention will greatly improve the specificity of such a β2-microglobulin detaining hemoperfusion device.

In a last aspect of the invention, a kit is provided which will simplify the use of the antibody embodiments of the invention for in vitro diagnostic purposes.

In addition to the antibody component, the kit may comprise reagents for preparing a suitable medium for carrying out an immunological reaction between the antibody and any des-Lys⁵⁸-β2-microglobulin present in a biological material and reagents allowing the detection of the antigen-antibody complexes formed. Reagents provided as parts of the kit comprise buffers, secondary antibodies, preparations of enzymes including enzyme conjugated antibodies, enzyme substrates and support materials.

With respect to the above description of the various aspects of the present invention and of the specific embodiments of these aspects it should be understood that any feature and characteristic described or mentioned above in connection with one aspect and/or one embodiment of an aspect of the invention also apply by analogy to any or all other aspects and/or embodiments of the invention described.

DESCRIPTION OF FIGURES

Figure 1. : Schematics of the cleavage of normal β2-microglobulin (A) to des-Lys⁵⁸-β2- microglobulin (C) through an activated complement Cls-cleaved intermediate (B) that is modified in vivo by a carboxypeptidase-activity present in plasma.

Figure 2. : Primary screening of supernatants for specific reactivity with des-Lys⁵⁸-β2- microglobulin. The clone marked with an X is specific for des-Lys⁵⁸-β2-microglobulin.

Figure 3. : Sample chromatogram from the purification of mouse immunoglobulin by affinity chromatography.

Figure 4. : ELISA test results for purified antibody 332-01 confirming specific binding of des-Lys⁵⁸-β2-microglobulin.

Figure 5. : Mass spectra from the analysis of β2-microglobulin and des-Lys⁵⁸-β2- microglobulin purified from urine.

Figure 6. : Graphical representation of the development of serum titers of antibodies against des-Lys⁵⁸-β2-microglobulin in peptide immunised mice determined by the use of a capture ELISA.

Figure 7. : Titration of mouse sera after the 4. bleed.

Figure 8. : Specific reactivity with des-Lys⁵⁸-β2-microglobulin of the three final clones 4-G3, 10-B5, and 13-D5.

Figure 9. : Characterisation of β2-microglobulin species in serum from dialysis patients as compared to normal serum by use of immunoaffinity-liquid chromatography-electrospray inonization mass spectrometry.

Figure 10. : Capillary electrophoresis of solution containing β2-microglobulin, des-Lys⁵⁸-β2- microglobulin and a marker protein after incubation with respectively immobilised monoclonal antibody against des-Lys⁵⁸-β2-microglobulin, β2-microglobulin and anti-β- amyloid as control.

Figure 11 : Capture ELISA for specific detection of des-Lys⁵⁸-β2-microglobulin in serum. Demonstration of specificity by analysis of normal serum with (dark columns) or without (grey columns) additions of 0.1 μg/ml of des-Lys⁵⁸-β2-microglobulin. Bars represent the standard deviation of duplicate experiments and shown are three sets of experiments carried out with different dilutions of the polyclonal rabbit anti-β2-microglobulin antibody as indicated

Figure 12: Measurement of des-Lys⁵⁸-β2-microglobulin levels in normal control sera (n= 17) and in sera from hemodialysis patients (n=25) using the capture ELISA based on the monoclonal anti-des-Lys⁵⁸-β2-microglobulin IgG antibodies (332-01). Horizontal lines indicate means.

EXAMPLES

Example 1

Purification of β -microqlobulin and des-Lvs⁵⁸-B2-microglobulin

The biological molecules β₂-microglobulin and des-Lys⁵⁸-β2-microglobulin were purified from urine exactly as described (Nissen MH et al., J. Immunol Methods 1997; 205: 29-33). The purified molecules were analyzed by electrospray-ionization mass spetrometry and showed the expected masses and an additional minor fraction of Met-99 oxidized species with a + 16 mass increase. Results from the analysis are shown in figure 5A. Mass spectrometry was performed on a Mariner electrospray ionization time-of-fligth analyzer using direct injection at 5 μl/min of a solution of approximately 1 mg/ml diluted 1 : 5 (vohvol) in 0.1% formic acid in 35% (vol/vol) acetonitrile in water. Electrospray settings were: positive ion mode at a spray tip potential of 4 kV and nozzle potential of 140 V with temperatures of 140°C in quadropole as well as in nozzle. Spectra were acquired at a frequency of 6 s per spectrum and the software-deconvoluted spectra are shown below together with the schematical theoretical structures in fig. 5B.

Example 2

Production of monoclonal antibodies

NMRI female mice (a group of five mice) were pre-immunized with BCG once and then 3 weeks after immunized intraperitoneally with tuberculin-coupled peptide of the sequence: tuberculin-Cys-Val-Glu-His-Ser-Asp-Leu-Ser-Phe-Ser-OH in a total amount of 25 μg peptide conjugate in 500 μl phosphate-buffered saline (PBS) with 1 mg AI(OH)₃. Peptide:conjugate molar ratio was 1:5 and 0.1% (vol/vol) glutaraldehyde in PBS was used for the peptide-carrier conjugation overnight at 4°C. After dialyzing the conjugated peptide against PBS using a 3,500 molecular weight cut-off dialysis membrane the mice were immunized 6 times at biweekly intervals and venous blood samples were obtained 10 days after each immunization. EDTA-plasma was tested for reactivity with same peptide coupled to ovalbumin at the N-terminal Cys-residue and coated onto the wells of microtiter plates (Maxisorb, Nunc, Roskilde, Denmark) as described in Example 3. After completing immunizations one mouse had a high titer response (se Example 3) and this mouse was subsequently sacrificed 3 days after boosting i.v. with 25 μg tuberculin-peptide conjugate. The spleen from the sacrificed mouse was removed and the spleen cells were fused with the HGPRT (hypoxanthine guanine phosphoribosyl transferase)-negative myeloma cell line P3X63Ag8.653 using polyethylene glycol. The cell fusion mixture was grown in a HGPRT+ selective medium, i.e. 10% fetal calf serum in DMEM containing hypoxantine, aminopterin, and thymidine (HAT) on a feeder cell layer of mouse macrophages in the wells of microtiter plates. After cell culture for 7 days at 37°C in air containing 6% C0₂ the medium was replaced and the cell cultures grown for additional 4 days under the same conditions before being subjected to screening for specific antibody reactivity with desLys-β₂m as described in Example 4. Figure 2 shows an example of the result of the primary screening. Only the cultures yielding the supernatants marked with X were selected for further processing. In all, a total of 730 hybridoma supernatants were tested and 4 of these cultures showed specificity towards desLys⁵⁸-β₂m.

Selected hybridoma cell cultures were further processed by limiting dilution using the test in Example 4 to ascertain specific reactivity and to ensure monoclonality. All three clones finally produced were isotyped using a typing kit from Amersham and were shown to be IgGj, ic-light chains.

Purification of monoclonal antibodies

The specific anti-desLysβ₂m antibody secreting clones were cultured in 5% C0₂ in air at 37°C for 2 weeks in RPMI1640 medium containing 15% fetal calf serum. The supernatant of these cells (approximately 2 liters from each of the three clones) contained mouse immunoglobulin that was purified by affinity chromatography on a protein G-Sepharose 4FF column connected to an automated liquid chromatography system (Phast-system, Amersham) as follows using a flow rate of 1 mL/min throughout:

(1) Portions of 200 mL culture supernatant was pumped onto an equilibrated 5 mL column of protein G Sepharose. (2) The column was washed until reaching a stable baseline when monitoring at 280 nm using phosphate-buffered saline, pH 7.4 (3) The bound immunoglobulin was then eluted using 0.1 M glycine/HCI, pH 2.8 in fractions of 1 mL into tubes containing 50 μL of 1 M Tris-base for pH-neutralization (4) The 280 nm absorbing eluate fractions were pooled and dialyzed against PBS at 4°C for 24 hours (see Figure) (5) The solution was concentrated by ultrafiltration on a molecular weight cut-off filter of 30,000 to a protein concentration of approximately 1 mg/mL as estimated by a bicinchonic acid assay (Pierce)

A sample chromatogram is shown in Figure 3.

The resulting purified monoclonal antibodies were tested for specific binding of desLysβ₂m in a capture ELISA as described in Example 4. Results for purified antibody 332-01 are shown in Figure 4.

Example 3

To show the development in serum titers of antibodies against β₂-microglobulin-peptyide in peptide-immunized mice a capture enzyme-linked immunosorbent assay was devised as follows: (1) An irradiated polystyrene microtiter plate (MaxiSorb from Nunc) was coated overnight in the cold with 1 μg/well of synthetic peptide H-Cys-Val-Glu-His-Ser- Asp-Leu-Ser-Phe-Ser-OH conjugated with ovalbumin through its N-terminal Cys- residue exactly as described for the peptide-tuberculin-conjugation in Example 2. The peptide was dissolved in coating buffer at 10 μg/mL. (2) After washing three times 5 minutes in washing buffer containing 0.05% (v/v) Tween 20 the plate was then blocked for 1 hour at room temperature with 2% (w/v) bovine serum albumin in washing buffer (8.0 g (137 mM) NaCI, 1.16 g (6.5 nM) Na₂HP0₄, 0.2 g (1.5 mM) KH₂P0₄, 0.2 g (2.7 mM) KCI, pH 7.4 to 1 L with water) on a shaker. (3) After washing three times as before the coated plate was incubated with dilutions of mouse sera (two-fold dilutions, starting at 1 : 2000 and ending at 1 : 128000) in washing buffer containing 0.05% (v/v) Tween 20 for 1 hour at room temperature on a shaker. (4) Plates were then washed as before and subsequently incubated with alkaline phosphatase-Iabelled goat anti-mouse immunoglobulin antibody (Sigma A-3688) diluted 1 :2000 in washing buffer with 0.05% Tween 20 for one hour at room temperature on a shaker. (5) After washing as before the alkaline phosphatase-activity was visualized using p- nitrophenyl phosphate tablets (Sigma S0942) and plate wells were read for absorbancies at 405 nm with signal at 690 nm subtracted. Figure 6 shows antibody response at different immunization dates at 1 :4000 serum dilutions and Figure 7 shows the titration of the mouse sera after the 4^th bleed. Mouse number 3 was chosen for the subsequent myeloma production.

Example 4

To screen for IgG antibodies for the des-Lys⁵⁸-β2-microglobulin in solution and show the preferential binding activity of the mouse monoclonal IgG antibodies a capture enzyme- linked immunosorbent assay was devised as follows:

(6) An irradiated polystyrene microtiter plate (MaxiSorb from Nunc) was coated overnight in the cold with 100 μL polyclonal rabbit anti-β₂-microglobulin antibody (DAKO A0072) diluted 1 : 1000 in coating buffer. (7) After washing three times 5 minutes in washing buffer containing 0.05% (v/v) Tween 20 the plate was then blocked for 1 hour at room temperature with 2% (w/v) bovine serum albumin in washing buffer (8.0 g (137 mM) NaCI, 1.16 g (6.5 nM) Na₂HP0₄, 0.2 g (1.5 mM) KH₂P0₄, 0.2 g (2.7 mM) KCI, pH 7.4 to 1 L with water) on a shaker. (8) After washing three times as before the plate coated with capture antibody was then incubated with washing buffer containing 0.05% (v/v) Tween 20 and 0.1 μg/ml of either β₂-microglobulin or des-Lys⁵⁸-β2-microglobulin for 1 hour at room temperature on a shaker. (9) After washing as before the plate was then incubated with 100 mikroliter/well of myeloma cell culture supernatant diluted 1: 10 in washing buffer with 0.05% Tween 20 for one hour at room temperature on a shaker. Every supernatant was tested in parallel on β₂-microglobulin and des-Lys⁵⁸-β2-microglobulin. (10) Plates were then washed as before and subsequently incubated with alkaline phosphatase-labelled goat anti-mouse immunoglobulin antibody (Sigma A-3688) diluted 1 :2000 in washing buffer with 0.05% Tween 20 for one hour at room temperature on a shaker. (11) After washing as before the alkaline phosphatase-activity was visualized using p- nitrophenyl phosphate tablets (Sigma S0942) and plate wells were read for absorbancies at 405 nm with signal at 690 nm subtracted.

The cell fusion yielded a total of 780 cell culture supernatants that were tested as described. Only supernatants containing mouse IgG that reacted specifically with des- Lys⁵⁸-β2-microgIobulin alone were chosen. Of the 4 supernatants that had this desired behavior 3 were succesfully cloned by limiting dilution. Figure 8 shows the specific reactivity of the final three clones (4-G3, 10-B5, and 13-D5).

Example 5

The state of β₂-microglobulin was examined by immunoaffinity-liquid chromatography- electrospray ionization mass spectrometry in sera from normal individuals and sera from patients in dialysis treatment. An example is shown in figure 9.

Eaxample 6

To show the specific binding activity of the mouse monoclonal IgG antibodies for the des- Lys⁵⁸-β2-microglobulin in solution capillary electrophoresis was performed as follows:

Monoclonal antibody 332-01 was bound to protein G Sepharose, that had previously been washed with electrophoresis buffer, by incubating 100 μL of the protein G slurry with 400 μL 332-01 (1.1 mg/mL in PBS) for 7 min at 25°C. After washing the protein G + 332-01 was incubated Vi h at 25°C with a mixture of 10 μL wt-β₂m (2 mg/mL) and 10 μL ΔK58- β₂m (2 mg/mL). The sample was spun down and capillary electrophoresis (CE) was done on the supernatant containing the unbound molecules. As a control protein G Sepharose bound to 1) a monoclonal antibody specific to both variants of β₂-microglobulin (anti-β₂m mAb) and 2) a monoclonal antibody that does not bind any of the variants (anti-β-amyloid mAb) were also incubated with a wt-β₂m - ΔK58-β₂m mixture. Again the sample was spun down and CE was performed on the supernatant. All the samples were mixed with 0.04 mg/ml marker peptide Acetyl-PSKD-OH.

The CE experiments was performed on a Beckman P/ACE 2050 instrument equipped with liquid sample cooling and UV-detection. Electrophoresis buffer was 0.1 M phosphate (0.081 M Na₂HP0₄ + 0.019 M NaH₂P0₄), pH 7.38 and samples were pressure injected for 8 s corresponding to a sample volume of 9 nL and electrophoresed at a constant current of 90 μA. Detection took place at 200 nm and the separation tube was a 50 μm inner diameter uncoated fused silica capillary (Polymicro Technologies or Beckman Coulter) of 47 cm total length with 40 cm to the detector window. Separations were typically carried out at 80-100 μA constant current corresponding to field strengths of 200-400 V/cm. Figure 10 shows that the monoclonal antibody against des-Lys⁵⁸-β2-microglobulin binds specifically hereto; it also shows that des-Lys⁵⁸-β2-microglobulin has two conformations (s- and f-) giving rise to two distinct peaks.

Example 7 To show quantitative and specific measurement of des-Lys⁵⁸-β2-microglobulin in serum using the anti-des-Lys⁵⁸-β2-microglobulin IgG monoclonal antibodies in solid-phase immunoassays capture ELISAs were performed as follows.

10 Maxisorb (NUNC) microtiter plate wells were coated overnight at 4°C with the monoclonal anti-des-Lys⁵⁸-β2-microglobulin antibody (hybridoma 332-01) at a concentration of 50 μg/ml in PBS. Blocking for 1 hour with 2% bovine serum albumin in PBS was followed by incubation for 1 hour with serum at 1/10 dilution. The plate was then incubated with a

15 1/1000 dilution of polyclonal, commercially available rabbit-anti-β₂m (DAKO) in PBS for 1 hour at room temperature. Subsequently, the plates were incubated with a 1/2000 dilution of alkaline phosphatase-conjugated goat-anti-mouse-antibody in PBS for 1 hour at room temperature and reactions were visualized using p-nitrophenyl phosphate and measurement of absorbance at 405 nm. The use of an internal standard made the results

20 comparable, and the des-Lys⁵⁸-β2-microglobulin serum concentration was calculated based on ELISA results of a healthy donor spiked with des-Lys⁵⁸-β2-microglobulin

Figure 11 illustrates the specificity of des-Lys⁵⁸-β2-microglobulin detection in serum using the capture ELISA outlined above with three different dilutions of the polyclonal rabbit anti- 25 β2-microglobulin as indicated. Serum from a healthy donor was spiked with purified des- Lys⁵⁸-β2-microglobulin at 0.1 μg/ml (blue columns) or measured without additions (grey columns). Shown are absorbance readings and the standard deviations of duplicate experiments.

30 Example 8 The capture ELISA based on the monoclonal anti-des-Lys⁵⁸-β2-microglobulin antibodies as described in Example 7 was used to ascertain des-Lys⁵⁸-β2-microglobulin levels in sera from hemodialysis (HD) patients and from normal controls. Figure 12 shows the results of

35 25 hemodialysis patients (9 female, 16 male (33 to 83 years of age)) compared with 17 controls. REFERENCES

Nissen, M. H., Johansen, B., Bjerrum, 0. J., J. Immunol Methods 1997; 205. 29-33 Sen, J. W., Bergen, R. H., and Heegaard, N. H. H., Anal. Chem. 2003; 75, 1196-1202. Tachibana, K et al, J.Immunol. Methods 1984, 75, 43-51.

Heegaard et al, J.Biochem.Chem. 2002, 277 (13), 11184-11189.

Claims

1. An antibody directed specifically against des-Lys⁵⁸-β2-microglobulin or a fragment of des-Lys-β2-microglobulin, said antibody being capable of forming a complex with des-

Lys⁵⁸-β2-microglobulin present in a non-immobilised form.

2. An antibody according to claim 1, wherein said antibody is capable of forming a complex with des-Lys⁵⁸-β2-microglobulin present in solution.

3. An antibody according to claim 1 or 2, wherein said antibody is obtained from an animal which has been with one or more polypeptides comprising the sequence of SEQ ID NO. : 1 and/or SEQ ID NO. : 2 or a part hereof.

4. An antibody according to claim 3, wherein said antibody is obtained from an animal which has been immunised with one or more polypeptides comprising a C-terminal and/or an N-terminal part of said sequences.

5. An antibody according to any of the preceding claims, wherein said antibody is a monoclonal antibody.

6. An antibody according to any of the preceding claims, wherein said antibody is an IgG class antibody.

7. An antibody according to any of the preceding claims, wherein said antibody is suitable for use in a capture immunoassay.

8. An in vitro diagnostic method for detection and/or quantification of des-Lys⁵⁸-β2- microglobulin present in a biological material, the method comprising the steps of contacting a sample of said biological material or an aliquot or fraction hereof with an antibody according to any of claims 1 to 7 and detecting any complexes of said antibody and des-Lys⁵⁸-β2-microglobulin formed.

9. An in vitro diagnostic method according to claim 8, wherein said biological material is a tissue or body fluid.

10. An in vitro diagnostic method according to claim 9, wherein said biological material is a biopsy or body fluid selected from the group comprising blood, serum saliva, urine, semen, spinal fluid, joint fluid, pleural exudate, ascites, dialysis fluid and lymphatic fluid.

11. An in vitro diagnostic method according to any of claims 8 to 10, wherein said complex of antibody and des-Lys⁵⁸-β2-microglobulin is detected in a solid phase immunoassay.

5 12. An in vitro diagnostic method according to any of claims 8 to 11, wherein said complex of antibody and des-Lys⁵⁸-β2-microglobulin is detected in a capture immunoassay.

13. An in vitro diagnostic method according to any of claims 8 to 12, wherein said complex of antibody and des-Lys⁵⁸-β2-microglobulin is detected in an ELISA.

10 14. An in vitro diagnostic method according to any of claims 8 to 13, wherein said complex of antibody and des-Lys⁵⁸-β2-microglobulin is formed in solution.

15. An in vitro diagnostic method according to any of claims 8 to 14, wherein said complex 15 of antibody and des-Lys⁵⁸-β2-microglobulin is detected by immunoaffinity chromatography.

16. An in vitro diagnostic method according to any of claims 8 to 10 and 15, wherein said complex of antibody and des-Lys⁵⁸-β2-microglobulin is detected by the use of mass spectrometry.

20 17. An in vitro diagnostic method according to any of claims 8 to 10 and any of claims 15 and 16 wherein said complex of antibody and des-Lys⁵⁸-β2-microglobulin is detected by the use of immunoaffinity-liquid chromatgraphy-electrospray ionization mass spectrometry.

25 18. An in vitro diagnostic method according to any of claims 8 to 17, wherein said biological material originates from a subject suffering from a disease or disorder selected from the group comprising kidney diseases, chronic inflammatory diseases, autoimmune diseases and cancer.

30 19. An in vitro diagnostic method according to any of claims 8 to 18, wherein said biological material originates from a subject undergoing hemodialysis.

20. An in vitro diagnostic method according to any of claims 8 to 20 for diagnosing an amyloid disease or susceptibility to an amyloid disease.

35 21. An in vitro diagnostic method according to claim 20, wherein said amyloid disease is selected from the group comprising primary and secondary amyloid diseases.

22. An in vitro diagnostic method according to claim 8 to 21 for diagnosing dialysis-related amyloidosis (DRA) in patients at risk before or after clinical symptoms (compressed median nerve (carpal tunnel syndrome), joint pain, and joint swelling in chronic dialysis patients) or for assessing the risk of developing early DRA including activation of the 5 complement system in conjunction with dialysis procedures.

23. A pharmaceutical composition comprising an antibody as described in any of claims 1 to 7.

10 24. A method of preventing or reducing the formation of amyloid deposits, the method comprising a step of contacting a biological material with an antibody as described in any of claims 1 to 7 in order to reduce the amount of free des-Lys⁵⁸-β2-microglobulin present in said biological material.

15 25. An antibody according to any of claims 1 to 7 for use as a medicament.

26. Use of an antibody according to any of claims 1 to 7 for the preparation of a medicament.

20 27. A method for generating an antibody according any of claims 1 to 7, said method comprising the steps of a) injecting into an animal a compound comprising a peptide of the sequence SEQ ID NO. :3 under conditions allowing the generation of antibodies, b) isolating spleen cells from said animal and fusing the cells with a myeloma cell 25 line, c) screening the resulting hybridoma cell lines for reactivity with des-Lys⁵⁸-β2- microglobulin.

28. A device for reducing the amount of des-Lys⁵⁸-β2-microglobulin present in a biological 30 material, the device comprising an antibody according to any of claims 1 to 7.

29. A device according to claim 27, further comprising means for detaining said antibody from said biological material.

35 30. A device according to claim 27 or 28, wherein said antibody is attached to a solid support.

31. A diagnostic kit comprising an antibody according to any of claims 1 to 7.