Means and methods for high throughput sugar spectroscopy of proteins

Abstract

A method of determining a sugar profile of a protein comprising (a) contacting a sample comprising the protein with a first bead having an antibody against the protein coupled thereto, thereby forming an antibody-protein complex, (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having, coupled thereto, (i) a label that amplifies the generated signal and (ii) a lectin, thereby forming an antibody-protein-lectin complex; and (c) determining the sugar spectrum of the protein. Also disclosed are methods for diagnosing cancer, autoimmune diseases, and inflammatory diseases, and kits for performing the methods disclosed herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to European Patent Application No. 22167920.2 filed on April 12, 2022, the contents of which are hereby incorporated by reference in their entirety for all purposes. Technical Field

[0003] The present invention discloses a method for determining the sugar profile of a protein, comprising (a) contacting a sample containing the protein with a first bead having an antibody against the protein coupled thereto, thereby forming an antibody-protein complex, (b) contacting the antibody-protein complex with one or more additional beads, each of which has (i) a label for amplifying the generated signal and (ii) a lectin coupled thereto, thereby forming an antibody-protein-lectin complex; and (c) determining the sugar profile of the protein. Also disclosed are methods for diagnosing cancer, autoimmune diseases and inflammatory diseases and kits for implementing the methods disclosed herein. Background Art

[0004] Polysaccharides are present on a variety of different proteins, where they have an impact on protein transport, stability and folding, ultimately changing the biochemical and biophysical properties of the protein. In addition, polysaccharides can mediate proteolysis patterns or directly mediate ligand-receptor interactions, oncogenic signal transduction, immune recognition, migration, and both cell-cell and cell-matrix adhesion. Therefore, specific polysaccharides can exert selective advantages on tumor cells. Therefore, the presence of specific polysaccharides or the presence of specific polysaccharides on specific proteins can be used as, for example, a biomarker for cancer diagnosis.

[0005] Glycan structures can be analyzed by using binding molecules that specifically bind to particular glycan structures. In addition to antibodies specific for glycan structures, lectins can also be used. Lectins are carbohydrate binding proteins that are highly specific for sugar groups of other molecules. These binding molecules can be used in assays such as enzyme-linked immunosorbent assays (ELISAs), enzyme-linked immunolectin assays (ELLAs), magnetic ELLAs (MELLAs) using optical, fluorescent or electrochemiluminescent readouts or parallel / multiple Luminex-like assays to analyze the presence or absence of specific glycan structures.

[0006] WO2019 / 185515 discloses a method for determining the sugar profile of a target protein, which includes enriching the target protein with an antibody, and then contacting the resulting complex with one or more lectins. Li et al. (2013), Clinical Chemistry, 59 (1): 315-324 also discloses a method for determining the sugar profile of a protein. Here, the target protein is enriched by an antibody coupled to beads and then contacted with a labeled lectin.

[0007] Carlstrom et al. (2018), Technical note. AlphaLISA Technology discloses a method for determining the sugar profile of an antibody, wherein the antibody is bound to a lectin, which is itself coupled to a bead (donor bead), and the antibody is further bound to protein G coupled to an AlphaLISA acceptor bead as a modification. Thus, each bead is conjugated to the antibody, and if the glycan binds to the lectin of the donor bead, the glycosylation state of the antibody can be detected. When there is an interaction, the donor bead is brought into proximity with the acceptor bead, and the excitation of the donor bead results in a luminescent signal from the acceptor bead (protein G AlphaLISA bead).

[0008] In addition, Chen Li et al. (2011), Electrophoresis, 32 (15): 2028-2035 discloses a method for analyzing protein glycosylation patterns using a protein-specific capture antibody coupled to beads. In this sandwich assay, only one bead is used - a fluorescently labeled lectin is used as a detection reagent. With necessary adjustments, this also applies to Jun Natsuki et al. (2005), Biotechnology and Bioengineering, 93 (2): 225-230. In these two prior art documents that comment on antibody-lectin assays as fairly reliable tools for glycosylation analysis, there is not even a motivation or inspiration to modify this antibody-lectin assay, because, for example, Chen Li et al. (2011) clearly emphasize that future research should be multiplexing with additional antibodies on beads, rather than changing the entire assay and using a second bead for detection.

[0009] However, there is still a continuing need for further improved methods for determining the sugar profile of proteins. In particular, these methods should, in particular for cancer-specific markers, have lower detection limits, higher detection sensitivity and selectivity, preferably with shorter analysis times. The present invention aims to address this need. Summary of the invention

[0010] This need is addressed by the subject matter as defined in the claims and embodiments described herein.

[0011] Therefore, the present invention relates to a method for determining the sugar profile of a protein, comprising

[0012] (a) contacting a sample containing the protein with first beads having antibodies against the protein coupled thereto,

[0013] Thus forming an antibody-protein complex,

[0014] (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label that amplifies the signal generated and (ii) a lectin,

[0015] thereby forming an antibody-protein-lectin complex; and

[0016] (c) determining the carbohydrate profile of the protein.

[0017] The method of the present invention may further comprise the step of (d) comparing the sugar profile of the protein with a reference sugar profile of the protein to determine whether the sugar profile of the protein deviates from the sugar profile of the reference sugar profile.

[0018] The method of the present invention may further comprise step (a') of enriching the antibody-glycoprotein complex prior to step (b) of contacting the antibody-glycoprotein complex with one or more additional beads.

[0019] The method of the present invention may further comprise, before step (c) determining the carbohydrate profile of the protein, step (b') enriching the antibody-protein-lectin complex.

[0020] Preferably, the protein is a cancer biomarker protein, an autoimmune disease biomarker protein, an inflammatory disease biomarker protein, or a neurodegenerative disease biomarker protein. Preferably, the protein is an autoimmune disease biomarker protein. Preferably, the protein is an inflammatory disease biomarker. Preferably, the protein is a cancer biomarker protein, more preferably an ovarian cancer biomarker protein, a breast cancer biomarker protein, a colorectal cancer biomarker protein, a pancreatic cancer biomarker protein, a prostate cancer biomarker protein, a thyroid cancer biomarker protein, a liver cancer biomarker protein, a lung cancer biomarker protein, a gastric cancer biomarker protein, a testicular cancer biomarker protein, or a bladder cancer biomarker protein. More preferably, the prostate cancer biomarker protein is β-binding globin, TIMP-1, PSA, fPSA, or tPSA. Preferably, the protein is a neurodegenerative disease biomarker protein, more preferably α-synuclein, tau-protein, or amyloid β protein and its isoforms.

[0021] Preferably, the lectin is specific for the following substances: core fucose, antenna fucose, N-linked oligosaccharides containing Fucα1-6GlcNAc-N-Asn, Fucα1-6 / 3GlcNAc, α-L-Fuc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Gal, Fucα1-6GlcNAc, Manβ1-4GlcNAcβ1-4GlcNAc, branched N-linked hexasaccharides, Manα1-3Man, α-D-Man, (GlcNAcβ1-4) 2-4 ,Galβ1-4GlcNAc, GlcNAcα1-4Galβ1-4GlcNAc, (GlcNAcβ1-4) 2-5, Neu5Ac (sialic acid), Galβ1-3GalNAc-serine / threonine, Galα1-3GalNAc, Galβ1-6Gal, Galβ1-4GlcNAc, Galβ1-3GalNAc, GalN Acα1-3GalNAc, GalNAcα1-3Gal, GalNAcα / β1-3 / 4Gal, α-GalNAc, GalNAcβ1-4Gal, GalNAcα1-3(Fucα1-2)Gal, G alNAcα1-2Gal, GalNAcα1-3GalNAc, GalNAcβ1-3 / 4Gal, GalNAc-Ser / Thr (Tn antigen), Galβ1-3GalNAc-Ser / Thr (T antigen), GalNAcβ1-4GlcNAc (LacdiNAc), α-2,3Neu5Ac (α2-3 linked sialic acid), α-2,6Neu5Ac (α2-6 linked sialic acid), α-2,8Neu5Ac (α2- 8-linked sialic acid), sialic acid (α-2,3Neu5Ac, α-2,6Neu5Ac or α-2,8Neu5Ac), Neu5Acα4 / 9-O-Ac-Neu5Ac, Neu5Acα2-3Galβ1-4Glc / GlcNAc, Neu5Acα2-6Gal / GalNAc, N-linked bi-antennary, N-linked tri / tetra-antennary, branched β1-6GlcNAc, Galα1-3(Fucα1-2)Galβ1-3 / 4GlcNAc Ac, Galβ1-3(Fucα1-4)GlcNAc, NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc, Fucα1-2Galβ1-3(Fucα1-4)GlcNAc, Galβ1-4(Fucα1-3)GlcNAc, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, high mannose, sialyl Lewis a (sialylated Le a ) antigen, sialyl Lewis x (sialylated Le x ) Antigen, Lewis x (Le x ) antigen, sialyl Tn antigen, sialyl T antigen, Lewis y (Le y ) antigen, sulfated core 1 glycan, Tn antigen, T antigen, core 2 glycan, Lewis a (Le a ) antigen, (GlcNAcβ1-4) n, β-D-GlcNAc, GalNAc, Gal-GlcNAc, GlcNAc, Galα1-3Gal, Galβ1-3GalNAc, α-Gal, α-GalNAc, (GlcNAc) n , branched chain (LacNAc) n .

[0022] The present invention may further comprise the method as defined elsewhere herein, wherein the protein is a cancer biomarker protein, and wherein a deviation of the sugar profile from a healthy sugar profile of the cancer biomarker protein indicates that the subject may be at risk for cancer or may have cancer.

[0023] The invention may further comprise the method as defined elsewhere herein, wherein the protein is an autoimmune disease biomarker protein, and wherein a deviation of the sugar profile from a healthy sugar profile of the autoimmune disease biomarker protein indicates that the subject may be at risk for or may have an autoimmune disease.

[0024] The present invention may further comprise the method as defined elsewhere herein, wherein the protein is an inflammatory disease biomarker protein, and wherein a deviation of the sugar profile from a healthy sugar profile of the inflammatory disease biomarker protein indicates that the subject may be at risk for or may have an inflammatory disease.

[0025] The present invention may further comprise the method as defined elsewhere herein, wherein the protein is a neurodegenerative disease biomarker protein, and wherein a deviation of the sugar profile from a healthy sugar profile of the neurodegenerative disease biomarker protein indicates that the subject may be at risk for or may have a neurodegenerative disease.

[0026] The present invention also relates to a kit for implementing the method of the present invention for diagnosing whether a subject may be at risk of cancer or may have cancer, the kit comprising antibodies specific to the cancer biomarker protein as defined herein and one or more lectins as defined herein.

[0027] The present invention also relates to a kit for implementing the method of the present invention for diagnosing whether a subject may be at risk of or may have an autoimmune disease, the kit comprising an antibody specific for an autoimmune disease biomarker protein that is IgG and one or more lectins as defined herein.

[0028] The present invention also relates to a kit for implementing the method of the present invention for diagnosing whether a subject may be at risk of or may have an inflammatory disease, the kit comprising antibodies specific to an inflammatory biomarker protein that is IgG, IgA or CRP and one or more lectins as defined herein.

[0029] The present invention also relates to a kit for carrying out a method for diagnosing whether a subject may be at risk of or may suffer from a neurodegenerative disease, the kit comprising antibodies specific for a neurodegenerative biomarker protein and one or more lectins as defined herein, the neurodegenerative biomarker protein being alpha-synuclein, tau protein or amyloid beta protein and isoforms thereof.

[0030] Preferably, said first beads and said further beads are contacted with said sample simultaneously.

[0031] Preferably, said further beads are contacted with said sample immediately after contacting said first beads with said sample.

[0032] Preferably, the first beads are contacted with the sample immediately after the second beads are contacted with the sample.

[0033] Preferably, during performance of the method of any preceding claim, the first bead and the further beads are in solution.

[0034] Preferably, the first bead and / or the further beads are made of glass, plastic, metal, agarose, latex, metal nano- or micro-particles, metal oxide nano- or micro-particles or a magnetic material.

[0035] Preferably, the label of the additional beads is an enzyme, a radioisotope, a fluorescent protein, a fluorescent dye, a bioluminescent marker or a tag (such as biotin).

[0036] The label of the one or more additional beads may be detected based on optical, fluorescent, luminescent, electrochemiluminescent and / or multi-analyte profiling (xMAP) readouts or means.

[0037] Preferably, for each of the one or more additional beads for each carbohydrate detected by the lectin, a different label is used in combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and accompanying drawings, in which:

[0039] Figure 1An exemplary scheme of an exemplary embodiment of the invention is depicted. A first bead (B1 ), further beads (B2) and the protein (analyte) are depicted.

[0040] Figure 2 Depicted is the assay configuration for high-throughput fPSA glycoprofiling (without any washing steps). In the first chamber, anti-fPSA modified magnetic nanoparticles (1) are mixed with a sample containing the analyte, i.e., fPSA (2), and then held at the bottom of the second chamber by a magnetic field. Through this second chamber, lectin / peroxidase modified nanoparticles (3) are first introduced and subsequently washed away by a substrate solution (4). After a short time, the solution is pumped into a spectrophotometer chamber (5) and the signal - color change - is detected. Inset: ROC curve showing the difference between tPSA (black line) and PGI+ index (light grey line) under this arrangement, with an AUC value of 0.686 (CI95% = [0.581; 0.781], specificity = 0.654, sensitivity = 0.690, precision = 0.673) for tPSA and 0.803 (CI95% = [0.707; 0.890], specificity = 0.712, sensitivity = 0.828, precision = 0.773) for PGI+. The total number of samples used in this study was 110 (58 BPH and 52 PCa patients). DETAILED DESCRIPTION

[0041] The present invention is described in detail below and will be further illustrated by the accompanying examples and drawings.

[0042] As described above, the present invention describes a method for determining the sugar profile of a (target) protein. The method can be described in the following exemplary embodiments (see also Figure 1 and 2 , or Example 1 or 2). First, a (target) protein ( Figure 1 The analyte in Figure 2 fPSA) sample with the first bead ( Figure 1 "B1" in Figure 2 The first bead is contacted with an antibody against the protein ("MNPs+Ab" in the above) and the first bead has an antibody against the protein coupled thereto. The protein is thereby captured on the first bead. The protein can (but need not be) be enriched, for example, by applying a magnet if the first bead is magnetic and washing away unbound protein. The first bead-antibody-protein complex is then combined with one or more additional beads ( Figure 1 In "B2", Figure 2In one embodiment, the first bead is a first bead-antibody-protein complex. The ... In Carlstrom et al. (2018) - a sandwich assay which is also clearly distinguished from the method of the present invention, i.e. the present invention uses antibodies against glycoproteins instead of protein G - there is no amplification effect due to coupling of the label to additional / second beads also containing lectins as described above according to the present invention. In Carlstrom et al. (2018), the detectable signal is only generated by the acceptor beads coupled to protein G (which beads also do not contain any lectins).

[0043] Although WO2019 / 185515 and Li et al. (2013), Clinical Chemistry, 59(1):315-324 disclose methods for determining sugar profiles, neither of them describes one or more additional beads, each of which has (i) a label and (ii) a lectin coupled thereto.

[0044] Therefore, the present invention relates to a method for determining the sugar profile of a protein, comprising

[0045] (a) contacting a sample containing the protein with first beads having antibodies against the protein coupled thereto,

[0046] Thus forming an antibody-protein complex,

[0047] (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label and (ii) a lectin,

[0048] thereby forming an antibody-protein-lectin complex; and

[0049] (c) determining the carbohydrate profile of the protein.

[0050] The term "glycoprofile of a protein" means the carbohydrate structure of a (target) protein, such as the composition and / or structure of covalently linked carbohydrates, such as the amount, presence or absence of covalently linked carbohydrates. The term "glycoprofile analysis" or "determination of a carbohydrate profile" means determining the carbohydrate structure (such as the composition and / or structure of covalently linked carbohydrates, such as the amount, presence or absence of covalently linked carbohydrates) on the (target) protein.

[0051] The method of the present invention can be used to determine whether a protein has a specific sugar profile, or in other words, can be used to determine whether a protein carries specific glycans. It can also be used to distinguish whether a protein carries glycans that indicate whether it is diseased or not. Therefore, a protein with a control sugar profile, i.e., a protein with a known sugar profile, can be used as a standard or positive control and compared with a signal obtained by, for example, the protein present in a sample (obtained from a subject). In the presence of a deviation, another sugar profile may be indicated. Therefore, the method of determining the sugar profile of a protein of the present invention may further include the step (d) of comparing the sugar profile of the protein with the control sugar profile of the protein to determine whether the sugar profile of the protein deviates from the sugar profile of the control sugar profile.

[0052] However, the present invention is not limited to detecting only one specific glycan structure, but can be used to detect two, three, four, five or more than five different glycan structures. In this case, "glycoprofile analysis" includes the determination of more than one, such as two, three, four, five or more than five different carbohydrate structures (such as the composition and / or structure of covalently linked carbohydrates, such as the amount, presence or absence of covalently linked carbohydrates) on the target protein. Advantageously, a different label is used for each different glycan structure to be analyzed. Therefore, a specific pairing of a specific lectin coupled to a specific additional bead and a specific label is provided. Different detection modes can also be combined, for example, based on fluorescent, luminescent or chemiluminescent labels that provide corresponding signals. Therefore, for each of the one or more additional beads for each carbohydrate detected by the lectin, a different label is preferably used in combination.

[0053] In the method of the present invention, the first bead and / or one or more additional beads may be enriched. This may be used to even further reduce the background signal. "Enrichment" as used herein describes a process of increasing the amount of beads / complexes / substances in a mixture. Therefore, the method of measuring the sugar spectrum of a protein of the present invention may further include, before contacting the antibody-glycoprotein complex with one or more additional beads in step (b), step (a') enriching the antibody-glycoprotein complex. In addition, the method of measuring the sugar spectrum of a protein of the present invention may further include, before step (c) measuring the sugar spectrum of the protein, step (b') enriching the antibody-protein-lectin complex. For clarity and to state only the obvious facts, the steps (a') and / or (b') may also be added to the method of the present invention for diagnosing whether a subject may be at risk of cancer or may suffer from cancer, for diagnosing whether a subject may be at risk of autoimmune disease or may suffer from autoimmune disease, or for diagnosing whether a subject may be at risk of inflammatory disease of the present invention or may suffer from inflammatory disease, or for diagnosing whether a subject may be at risk of neurodegenerative disease or may suffer from neurodegenerative disease.

[0054] The robustness of the present invention allows the first bead and the additional beads to be contacted with the sample simultaneously or in immediate succession. Thus, the first bead and the additional beads may be contacted with the sample simultaneously. Alternatively, the additional beads may be contacted with the sample immediately after the first bead is contacted with the sample. Alternatively, the first bead may be contacted with the sample immediately after the second bead is contacted with the sample. This also means that the order of steps (a) and (b) need not be determined by the naming of steps (a) and (b). However, it is preferred that step (a) is followed by step (b) and then step (c).

[0055] In addition, preferably, during the implementation of the method described herein, the first bead and the additional beads are in solution. "In solution" in this context means that the first bead and the additional beads are not particularly fixed by magnetic force, nor are they coupled to a solid material such as a microwell plate, a column or a reaction tube. In one embodiment, the first bead is neither fixed by magnetic force nor coupled to a solid material such as a microwell plate, a column or a reaction tube. In one embodiment, the one or more additional beads are neither fixed by magnetic force nor coupled to a solid material such as a microwell plate, a column or a reaction tube.

[0056] There is no particular limitation on the protein (to be subjected to sugar profile analysis by the method of the present invention). However, preferably, the protein is a glycoprotein. Since the presence or absence of a specific glycan structure on a protein may be important for the diagnosis or prediction of a disease, preferably, the target protein is a protein whose sugar profile is associated with a disease. As used herein, the term "glycoprotein" (or "glycosylated protein") means a protein containing one or more N-, O-, S- or C-covalently linked carbohydrates of various types, for example, from monosaccharides to branched oligosaccharides or polysaccharides (including their modified forms, such as sulfo- or phosphate group attachment). N-linked glycans are -NH to asparagine. 2 O-linked glycans are carbohydrates that are bound to the -OH group of serine, threonine, or hydroxylated amino acids. S-linked glycans are carbohydrates that are bound to the –SH group of cysteine. C-linked glycans are carbohydrates that are bound to tryptophan via a C-C bond.

[0057] The term "carbohydrate" means a carbohydrate having the stoichiometric formula C n (H 2 O) n The general term "carbohydrate" includes monosaccharides, oligosaccharides and polysaccharides and substances derived from monosaccharides by reduction of the carbonyl group (sugar alcohol), oxidation of one or more terminal groups to carboxylic acids, or replacement of one or more hydroxyl groups with a hydrogen atom, an amino group, a thiol group or the like. Carbohydrates also include derivatives of these compounds.

[0058] As described herein, the presence of a specific sugar profile or glycan structure on a target protein may be relevant to the diagnosis of a specific disease such as cancer, autoimmune disease, inflammatory disease or neurodegenerative disease. Some combinations of known (target) proteins (i.e., biomarker proteins) and glycan structures (A) indicate diseases. Error! The reference source was not found (Fehler! Verweisquelle konnte nicht gefunden warden), which exemplarily lists specific combinations of (target) proteins and glycans indicating diseases and antibodies or lectins that bind to specific glycan structures. Therefore, the methods and uses of the present invention can be used to diagnose diseases such as cancer, autoimmune disease, inflammatory disease or neurodegenerative disease. Therefore, the presence of the specific sugar profile or glycan structure can indicate diseases such as cancer, autoimmune disease, inflammatory disease or neurodegenerative disease.

[0059] In the present context, the protein is preferably a cancer biomarker protein, an autoimmune disease biomarker protein, an inflammatory disease biomarker protein or a neurodegenerative disease biomarker protein. More preferably, the protein is preferably a cancer biomarker protein. More preferably, the protein is preferably an autoimmune disease biomarker protein. More preferably, the protein is preferably an inflammatory disease biomarker protein. More preferably, the protein is preferably a neurodegenerative disease biomarker protein.

[0060] As used herein, "autoimmune disease" refers to a group of diseases characterized by the production of antibodies against one's own tissues. Non-limiting examples of autoimmune diseases include, but are not limited to, Hashimoto's disease, primary biliary cirrhosis, systemic lupus erythematosus, rheumatic fever, rheumatoid arthritis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, and post-viral encephalomyelitis, Adson's disease, autoimmune bowel disease, primary biliary cirrhosis, Goodpasture's syndrome, Hashimoto's thyroiditis, myasthenia gravis, myxedema, pemphigoid, rheumatoid arthritis, Sjogren's syndrome, symphathetic ophthalmitis, two forms of lupus erythematosus, thyrotoxicosis, ulcerative colitis, multiple sclerosis, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, and psoriasis.

[0061] As used herein, "inflammatory disease" refers to a group of diseases characterized by damage and / or abnormal functioning of the body's inflammatory mechanisms. Non-limiting examples of inflammatory diseases include, but are not limited to, necrotizing enterocolitis, gastroenteritis, pelvic inflammatory disease (PID), empyema, pleurisy, pyelitis, pharyngitis, angina, arthritis, acne, urinary tract infection, acne vulgaris, asthma, celiac disease, chronic prostatitis, colitis, diverticulitis, glomerulonephritis, hidradenitis pustularis, allergies, inflammatory bowel disease, interstitial cystitis, mast cell activation syndrome, mastocytosis, otitis, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, rhinitis, sarcoidosis, transplant rejection, vasculitis.

[0062] As used herein, "neurodegenerative disease" refers to a group of diseases characterized by impaired and / or abnormal brain function. Non-limiting examples of inflammatory diseases include Parkinson's disease, Alzheimer's disease and other forms of tauopathy, primary age-related tauopathy (tautopathy), chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, chromosome 17-related frontotemporal dementia with Parkinson's syndrome, vacuolar tauopathy, Lytico-bodig disease, ganglioglioma and gangliocytoma, meningioangiomatosis, post-encephalitis Parkinson's syndrome, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, pantothenate kinase-related neurodegeneration and lipofuscinosis, etc.

[0063] As used herein, "cancer" refers to a group of diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream. Non-limiting examples of cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, kidney cancer (such as clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (such as renal cell carcinoma (RCC)), prostate cancer (such as hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer (stomach cancer), bladder cancer, hepatoma, breast cancer, colon cancer and head and neck cancer (or tumor), gastric cancer (gastric cancer). cancer), germ cell tumors, pediatric sarcomas, sinonasal natural killer, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors of childhood, ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, virus-related cancers (e.g., human papillomavirus (HPV)-related tumors), and hematological malignancies derived from either of the two major blood cell lineages (the two major Blood cell lineages, i.e., myeloid cell lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lineage (which produces B, T, NK, and plasma cells), such as all types of leukemias, lymphomas, and myelomas, such as acute, chronic, lymphocytic, and / or myeloid leukemias, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or eosinophilic M4 variant [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroma;Lymphomas, such as Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant, lymphoproliferative disorders, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematologic neoplasms of the lymphoid lineage, acute myeloid leukemia, hematologic neoplasms of the lymphoid lineage ... Lymphocytic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), and leukemia. myeloma), solitary, plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hair cell lymphoma; myeloid blood cell tumors, mesenchymal origin tumors, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma, central and peripheral nerve tumors, including astrocytoma, schwannoma; mesenchymal origin tumors, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratoma, lymphoid lineage blood cell tumors, such as T cell and B cell tumors, including but not limited to T cell diseases, such as T-prolymphocytic leukemia (T-PLL), including small cell and brain cell types; large granular lymphocytic leukemia (LGL), preferably T cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (polymorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head or neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of the above cancers. Preferred cancers are also shown in Error! Reference source not found. ;

[0064] The cancer may also be ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, prostate cancer, thyroid cancer, liver cancer, lung cancer, stomach cancer, testicular cancer or bladder cancer. Thus, the (target) biomarker protein may be an ovarian cancer biomarker protein, a breast cancer biomarker protein, a colorectal cancer biomarker protein, a pancreatic cancer biomarker protein, a prostate cancer biomarker protein, a thyroid cancer biomarker protein, a liver cancer biomarker protein, a lung cancer biomarker protein, a stomach cancer biomarker protein, a testicular cancer biomarker protein or a bladder cancer biomarker protein.

[0065] Exemplary cancers, cancer biomarkers with aberrant glycosylation, lectins, antibodies and corresponding glycan modifications within the meaning of the present invention are also shown below Error! Reference source not found. Error! Lectin abbreviations used in the referenced sources not found: AAA – Anguilla anguilla agglutinin (UniProtKB accession number: Q7SIC1), AAL – Aleuria aurantia lectin, ABA – Agaricus bisporus agglutinin, ACA – Amaranthus caudatus agglutinin, AHA – Arachis hypogaea agglutinin = peanut agglutinin (PNA), AIA – Artocarpus integrifolia agglutinin = Jacalin, AlloA – Allomyrina dichotoma agglutinin, AOL – Aspergillus oryzae agglutinin lectin), BanLec–Musa paradisiaca lectin, BS-I–Bandeiraea simplicifolia lectin = Griffonia (Bandeiraea) simplicifolia lectin I, Con A–Concanavalin A, DBA–Dolichos biflorus agglutinin, DSA–Daturastramonium agglutinin Jacalin, ECL–Erythrinacristagalli lectin, GNA–Galanthus nivalis agglutinin, GSAI (GSLI)–Griffonia (Bandeiraea) simplicifolia lectin I, GSL II – Griffonia (Bandeiraea) simplicifolia lectin II, HHL – Hippeastrumhybrid(Amaryllis)lectin), HPA–Helixpomatia agglutinin, LBA–Phaseolus lunatus(lima bean, LBA), LEL–Lycopersicon esculentum(tomato)lectin), LCA–Lens culinaris agglutinin, LTA–Lotus tetragonolobuslectin, MAA I–Maackia amurensis agglutinin I, MAA II–Maackia amurensis agglutinin II, MGBL 1–macrophage-galactose-binding lectin 1, MGBL2 (macrophage-galactose-binding lectin 2), NPA–Narcissus lectin pseudonarcissus (Daffodil) lectin), PHA E– Phaseolus vulgaris agglutinin E, PHAL– Phaseolus vulgaris agglutinin L, PhoSL– Pholiotasquarrosa lectin, PNA– Peanut agglutinin, PSL– Pisumsativum lectin, PTAI– Psophocarpus tetragonolobus lectin I, PTAII– Psophocarpus tetragonolobus II, PWM– Phytolacca americana, RCA I– Ricinus communis agglutinin I, RCAII– Ricinus communis agglutinin II II), SBA – Soybean agglutinin (soybean agglutinin (Glycine max agglutinin)), SCA – Sambucus canadensis agglutinin = Sambucus nigraagglutinin (SNA), SJA – Sophora japonica agglutinin II, SNA – Sambucus nigra agglutinin, SSA – Sambucus sieboldiana agglutinin, SSL – Salvia sclarea lectin, STL – Solanum tuberosum lectin, TJA-I – Trichosanthes japonica agglutinin I, TJA-II – Trichosanthes japonica agglutinin (Yamashita et al.), TVA – Triticum vulgaris agglutinin = WGA – wheat germ agglutinin, UEA – Ulex europaeus agglutinin =Agglutinin, VVA – Viciavillosa lectin, WFA – Wisteria floribunda lectin, WGA – wheat germ agglutinin = TVA – Triticum vulgaris agglutinin. The symbol “↑”, an arrow pointing upward, indicates an increase in the concentration of the corresponding glycan or complex (e.g., dimer, trimer, etc.). The symbol “↓”, an arrow pointing downward, indicates an increase in the concentration of the corresponding glycan or complex (e.g., dimer, trimer, etc.).

[0066] Table 1: Cancers, corresponding cancer biomarkers with aberrant glycosylation, lectins and antibodies. The combinations in this table are only examples of different cancer types. The present invention is not limited to these exemplary combinations.

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[75] N. Yang, S. Feng, K. Shedden,

[0166]

[76] Yamashita K, Umetsu K, Suzuki T, Ohkura T (1992) Purification and characterization of a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc and HSO3(-)-->6Gal beta 1-->GlcNAc specific lectin in tuberous roots of Trichosanthesjaponica.Biochemistry 31(46):11647-11650.

[0167] The method of determining the sugar profile of a protein of the present invention provides information about the sugar profile of the protein. This information can be used to diagnose various diseases as described herein (see also Table 1). Diseases known to be characterized by proteins with altered sugar profiles include, but are not limited to, cancer, autoimmune diseases, inflammatory diseases, or neurodegenerative diseases.

[0168] Therefore, the present invention also relates to a method for diagnosing whether a subject may be at risk of cancer or may have cancer, comprising

[0169] (a) contacting a sample obtained from the subject and containing a cancer biomarker protein with first beads having antibodies against the cancer biomarker protein coupled thereto,

[0170] Thus forming an antibody-protein complex,

[0171] (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label and (ii) a lectin,

[0172] thereby forming an antibody-protein-lectin complex; and

[0173] (c) determining the sugar profile of the cancer biomarker protein,

[0174] Wherein a deviation of the sugar profile from a healthy sugar profile of the cancer biomarker protein indicates that the subject may be at risk for cancer or may have cancer.

[0175] Therefore, the present invention further relates to a method for diagnosing whether a subject may be at risk of or may have an autoimmune disease, comprising

[0176] (a) contacting a sample obtained from the subject and containing an autoimmune disease biomarker protein with first beads having antibodies against the autoimmune disease biomarker protein coupled thereto,

[0177] Thus forming an antibody-protein complex,

[0178] (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label and (ii) a lectin,

[0179] thereby forming an antibody-protein-lectin complex; and

[0180] (c) determining the sugar profile of the autoimmune disease biomarker protein,

[0181] Wherein a deviation of the sugar profile from a healthy sugar profile of the autoimmune disease biomarker protein indicates that the subject may be at risk for or may have an autoimmune disease.

[0182] In the case of autoimmune disease, a carbohydrate profile indicating that the subject may be at risk for or may have an autoimmune disease may include increased desialylation, or exposure of GlcNAc and mannose on antibodies, which may lead to activation of the "alternative lectin pathway of complement activation" by MBP (mannose binding protein).

[0183] Therefore, the present invention also relates to a method for diagnosing whether a subject may be at risk of or may have an inflammatory disease, comprising

[0184] (a) contacting a sample obtained from the subject and containing an inflammatory disease biomarker protein with first beads having antibodies against the inflammatory disease biomarker protein coupled thereto,

[0185] Thus forming an antibody-protein complex,

[0186] (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label and (ii) a lectin,

[0187] thereby forming an antibody-protein-lectin complex; and

[0188] (c) determining the carbohydrate profile of the inflammatory disease biomarker protein,

[0189] wherein a deviation of the sugar profile from a healthy sugar profile of the inflammatory disease biomarker protein indicates that the subject may be at risk for or may have an inflammatory disease.

[0190] Therefore, the present invention also relates to a method for diagnosing whether a subject may be at risk of or may suffer from a neurodegenerative disease, comprising

[0191] (a) contacting a sample obtained from the subject and containing a neurodegenerative disease biomarker protein with first beads having antibodies against the neurodegenerative disease biomarker protein coupled thereto,

[0192] Thus forming an antibody-protein complex,

[0193] (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label and (ii) a lectin,

[0194] thereby forming an antibody-protein-lectin complex; and

[0195] (c) determining the sugar profile of the neurodegenerative disease biomarker protein,

[0196] Wherein a deviation of the sugar profile from a healthy sugar profile of the neurodegenerative disease biomarker protein indicates that the subject may be at risk for or may have a neurodegenerative disease.

[0197] A "healthy sugar profile" of a biomarker protein describes the sugar profile of that biomarker in a sample obtained from a healthy (human) subject, who preferably does not have cancer, an autoimmune disease, a neurodegenerative disease, or an inflammatory disease, or any other specific disease associated with cancer, an autoimmune disease, an inflammatory disease, or a neurodegenerative disease as described herein.

[0198] The term "lectin" as used herein refers to a carbohydrate binding protein. Lectins are generally highly specific for one or more carbohydrate moieties (e.g., they react specifically with terminal glycoside residues of other molecules, such as one or more glycans of a glycoprotein (e.g., a branched sugar molecule of a glycoprotein, such as a target polypeptide within the meaning of the present invention and a biomarker as described in Table 1 herein). Lectins are well known in the art. A skilled person can readily determine which lectin can be used to bind to one or more target carbohydrate moieties, such as one or more carbohydrate moieties of a glycan attached to a protein. Preferred lectins for use in the context of the present invention are described herein. The term "lectin" also includes Siglecs (immunoglobulin-like lectins that bind sialic acid), galectins (lectins that specifically bind to glycans containing β-galactosyl) and selectins (lectins that bind to sialyl Lewis X (SLe x ) determinant NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc and related sialylated fucosylated glycans). It is worth noting that when used herein, the term "lectin" also refers to a glycan binding antibody. Therefore, when used herein, the term "lectin" may also include lectins, Siglecs, galectins, selectins, etc., as well as glycan binding antibodies. Lectins may also include DNA / RNA aptamers that recognize glycans.

[0199] Lectins can be specific for the following: core fucose, antennal fucose, N-linked oligosaccharides containing Fucα1-6GlcNAc-N-Asn, Fucα1-6 / 3GlcNAc, α-L-Fuc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Gal, Fucα1-6GlcNAc, Manβ1-4GlcNAcβ1-4GlcNAc, branched N-linked hexasaccharides, Manα1-3Man, α-D-Man, (GlcNAcβ1-4) 2-4 ,Galβ1-4GlcNAc, GlcNAcα1-4Galβ1-4GlcNAc, (GlcNAcβ1-4) 2-5, Neu5Ac (sialic acid), Galβ1-3GalNAc-serine / threonine, Galα1-3GalNAc, Galβ1-6Gal, Galβ1-4GlcNAc, Galβ1-3GalNAc, GalN Acα1-3GalNAc, GalNAcα1-3Gal, GalNAcα / β1-3 / 4Gal, α-GalNAc, GalNAcβ1-4Gal, GalNAcα1-3(Fucα1-2)Gal, G alNAcα1-2Gal, GalNAcα1-3GalNAc, GalNAcβ1-3 / 4Gal, GalNAc-Ser / Thr (Tn antigen), Galβ1-3GalNAc-Ser / Thr (T antigen), GalNAcβ1-4GlcNAc (LacdiNAc), α-2,3Neu5Ac (α2-3 linked sialic acid), α-2,6Neu5Ac (α2-6 linked sialic acid), α-2,8Neu5Ac (α2-8 linked sialic acid), sialic acid (α-2,3Neu5Ac, α-2,6Neu5Ac or α-2,8Neu5Ac), Neu5Acα4 / 9-O-Ac-Neu5Ac, Neu5Acα2-3Galβ1-4Glc / GlcNAc, Neu5Acα2-6Gal / GalNAc, N-linked bi-antennary, N-linked tri / tetra-antennary, branched β1-6GlcNAc, Galα1-3(Fucα1-2)Galβ1-3 / 4GlcNAc Ac, Galβ1-3(Fucα1-4)GlcNAc, NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc, Fucα1-2Galβ1-3(Fucα1-4)GlcNAc, Galβ1-4(Fucα1-3)GlcNAc, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, high mannose, sialyl Lewis a (sialylated Le a ) antigen, sialyl Lewis x (sialylated Le x ) Antigen, Lewis x (Le x ) antigen, sialyl Tn antigen, sialyl T antigen, Lewis y (Le y ) antigen, sulfated core 1 glycan, Tn antigen, T antigen, core 2 glycan, Lewis a (Le a ) antigen, (GlcNAcβ1-4) n, β-D-GlcNAc, GalNAc, Gal-GlcNAc, GlcNAc, Galα1-3Gal, Galβ1-3GalNAc, α-Gal, α-GalNAc, (GlcNAc) n , branched chain (LacNAc) n .

[0200] Carbohydrate abbreviations used herein include: "Neu5Ac" for N-acetylneuraminic acid; "Fuc" for fucose, "GalNAc" for N-acetylgalactosamine; "GlcNAc" for N-acetylglucosamine; "Gal" for galactose (e.g., Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, E.M.E., Essentials of Glycobiology, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY), 2009).

[0201] Additionally, as used herein, the following terms are defined as follows:

[0202] "Core fucose" means fucose linked to the C6 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom.

[0203] "Antennae fucose" means fucose linked to the C3 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom, or fucose linked to the C2 atom of an adjacent fucose via an α-glycosidic bond at its C1 atom,

[0204] "N-linked oligosaccharide containing Fucα1-6GlcNAc-N-Asn" means an oligosaccharide having fucose linked via an α-glycosidic bond of its C1 atom to the C6 atom of N-acetylglucosamine linked via an N-glycosidic bond to asparagine,

[0205] "Fucα1-6 / 3GlcNAc" means that fucose is linked to the C6 (C3) atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom,

[0206] "α-L-Fuc" means α-L-fucose,

[0207] "Fucα1-2Galβ1-4(Fucα1-3)GlcNAc" means that fucose is linked to the C2 atom of galactose via an α-glycosidic bond at its C1 atom, and the galactose is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom; meanwhile, a second fucose is linked to the C3 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom,

[0208] "Fucα1-2Gal" means that fucose is linked to the C2 atom of galactose via an α-glycosidic bond at its C1 atom.

[0209] "Fucα1-6GlcNAc" means that fucose is linked to the C6 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom,

[0210] “Manβ1-4GlcNAcβ1-4GlcNAc” means that mannose is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom, and the N-acetylglucosamine is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom,

[0211] "Branched N-linked hexasaccharide" means a non-linear glycan consisting of several sugars linked to asparagine via N-glycosidic bonds

[0212] "Manα1-3Man" means that mannose is linked to the C3 atom of mannose via an α-glycosidic bond at its C1 atom,

[0213] "α-D-Man" refers to α-D-mannose,

[0214] "(GlcNAcβ1-4) 2-4 " means that N-acetylglucosamine is repeatedly linked to the C4 atom of N-acetylglucosamine through a β-glycosidic bond of its C1 atom,

[0215] "Galβ1-4GlcNAc" means that galactose is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom,

[0216] "GlcNAcα1-4Galβ1-4GlcNAc" means that N-acetylglucosamine is linked to the C4 atom of galactose via an α-glycosidic bond at its C1 atom, and the galactose is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom,

[0217] "N-acetylglucosamine" means the amide between glucosamine and acetic acid,

[0218] "(GlcNAcβ1-4) 2-5" means that N-acetylglucosamine is repeatedly linked to the C4 atom of N-acetylglucosamine through a β-glycosidic bond of its C1 atom,

[0219] "Neu5Ac" (or sialic acid) means N-acetylneuraminic acid,

[0220] "Galβ1-3GalNAc-serine / threonine" means that galactose is linked to the C3 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom, and the N-acetylglucosamine is linked to serine / threonine,

[0221] "Galα1-3GalNAc" means that galactose is linked to the C3 atom of N-acetylgalactosamine via an α-glycosidic bond at its C1 atom.

[0222] "Galβ1-6Gal" means that galactose is linked to the C6 atom of galactose via a β-glycosidic bond at its C1 atom,

[0223] "Galβ1-4GlcNAc" means that galactose is linked to the C3 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom,

[0224] "Galβ1-3GalNAc" means that galactose is linked to the C3 atom of N-acetylgalactosamine via a β-glycosidic bond at its C1 atom,

[0225] "GalNAcα1-3GalNAc" means that N-acetylgalactosamine is linked to the C3 atom of N-acetylgalactosamine via an α-glycosidic bond at its C1 atom,

[0226] "GalNAcα1-3Gal" means that N-acetylgalactosamine is linked to the C3 atom of galactose via an α-glycosidic bond at its C1 atom,

[0227] "GalNAcα / β1-3 / 4Gal" means that N-acetylgalactosamine is linked to the C3 or C4 atom of galactose via an α- or β-glycosidic bond at its C1 atom,

[0228] "α-GalNAc" means the amide between α-galactosamine and acetic acid,

[0229] "GalNAcβ1-4Gal" means that N-acetylgalactosamine is linked to the C4 atom of galactose via a β-glycosidic bond at its C1 atom,

[0230] "GalNAcα1-3(Fucα1-2)Gal" means that N-acetylgalactosamine is linked to the C3 atom of galactose via an α-glycosidic bond at its C1 atom, and fucose is linked to the C2 atom of galactose via an α-glycosidic bond at its C1 atom,

[0231] "GalNAcα1-2Gal" means that N-acetylgalactosamine is linked to the C3 atom of galactose via an α-glycosidic bond at its C1 atom,

[0232] "GalNAcα1-3GalNAc" means that N-acetylgalactosamine is linked to the C3 atom of N-acetylgalactosamine via an α-glycosidic bond at its C1 atom,

[0233] "GalNAcβ1-3 / 4Gal" means that N-acetylgalactosamine is linked to the C3 or C4 atom of galactose via a β-glycosidic bond at its C1 atom,

[0234] "GalNAc-Ser / Thr" (or Tn antigen) means N-acetylgalactosamine linked to serine / threonine via an O-glycosidic bond,

[0235] "Galβ1-3GalNAc-Ser / Thr" (T antigen or Thomsen-Friedenreich antigen) means that galactose is linked to the C3 atom of N-acetylgalactosamine via a β-glycosidic bond at its C1 atom, and the N-acetylgalactosamine is linked to serine / threonine via an O-glycosidic bond,

[0236] "GalNAcβ1-4GlcNAc" (or LacdiNAc) means that N-acetylgalactosamine is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom,

[0237] "α2-3Neu5Ac" (or α2-3-linked sialic acid) means N-acetylneuraminic acid is linked to the C3 atom of the adjacent sugar via an α-glycosidic bond at its C2 atom.

[0238] "α2-6Neu5Ac" (or α2-6-linked sialic acid) means N-acetylneuraminic acid is linked to the C6 atom of the adjacent sugar via an α-glycosidic bond at its C2 atom.

[0239] "α2-8Neu5Ac" (or α2-8-linked sialic acid) means that N-acetylneuraminic acid is linked to the C8 atom of the adjacent N-acetylneuraminic acid via an α-glycosidic bond at its C2 atom,

[0240] "Neu5Acα4 / 9-O-Ac-Neu5Ac" means that N-acetylneuraminic acid is linked to the C9 atom of the adjacent O-acetyl N-acetylneuraminic acid via an α-glycosidic bond at its C4 atom.

[0241] "Neu5Acα2-3Galβ1-4Glc / GlcNAc" means that N-acetylneuraminic acid is linked to the C3 atom of galactose via an α-glycosidic bond at its C2 atom, and the galactose is linked to the C4 atom of glucose or N-acetylglucosamine via a β-glycosidic bond at its C1 atom,

[0242] "Neu5Acα2-6Gal / GalNAc" means that N-acetylneuraminic acid is linked to the C6 atom of galactose or N-acetylgalactosamine via an α-glycosidic bond at its C2 atom.

[0243] "N-linked biantennary" means a non-linear glycan with two antennae (sugar chains) linked to asparagine by N-glycosidic bonds,

[0244] "N-linked tri / tetra-antennary" means a non-linear glycan with three / four antennae (sugar chains) linked to asparagine via N-glycosidic bonds,

[0245] "Branched β1-6GlcNAc" means that N-acetylglucosamine is linked to the C6 atom of the adjacent sugar via a β-glycosidic bond at its C1 atom.

[0246] "Galα1-3(Fucα1-2)Galβ1-3 / 4GlcNAc" means that galactose is linked to the C3 atom of galactose via an α-glycosidic bond at its C1 atom, and the galactose is linked to the C3 or C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom; and fucose is linked to the C2 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom,

[0247] "Galβ1-3(Fucα1-4)GlcNAc" means that galactose is linked to the C3 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom; and fucose is linked to the C4 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom.

[0248] "NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc" means that N-acetylneuraminic acid is linked to the C3 atom of galactose via an α-glycosidic bond at its C2 atom, and the galactose is linked to the C3 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom; while fucose is linked to the C4 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom,

[0249] "Fucα1-2Galβ1-3(Fucα1-4)GlcNAc" means that fucose is linked to the C2 atom of galactose via an α-glycosidic bond at its C1 atom, and the galactose is linked to the C3 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom; meanwhile, a second fucose is linked to the C4 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom,

[0250] "Galβ1-4(Fucα1-3)GlcNAc" means that galactose is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom; and fucose is linked to the C3 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom.

[0251] "NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc" means that N-acetylneuraminic acid is linked to the C3 atom of galactose via an α-glycosidic bond at its C2 atom, and the galactose is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom; while fucose is linked to the C3 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom,

[0252] "Fucα1-2Galβ1-4(Fucα1-3)GlcNAc" means that fucose is linked to the C2 atom of galactose via an α-glycosidic bond at its C1 atom, and the galactose is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom; meanwhile, a second fucose is linked to the C3 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom,

[0253] "High mannose" means a polysaccharide containing more than three mannose units.

[0254] "Sialyl Lewis a "(sialylated Le a ) antigen is Neu5Acα2-3 / 6Galβ1-3(Fucα1-4)GlcNAc, which means that N-acetylneuraminic acid is linked to the C3 or C6 atom of galactose via an α-glycosidic bond at its C2 atom, and the galactose is linked to the C3 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom; and fucose is linked to the C4 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom,

[0255] "Sialyl Lewis x "(sialylated Le x) antigen is Neu5Acα2-3 / 6Galβ1-4(Fucα1-3)GlcNAc, which means that N-acetylneuraminic acid is linked to the C3 or C6 atom of galactose via an α-glycosidic bond at its C2 atom, and the galactose is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom; and fucose is linked to the C3 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom,

[0256] "Louis x ”(Le x ) antigen is "Galβ1-4(Fucα1-3)GlcNAc", which means that galactose is linked to the C4 atom of N-acetylglucosamine through a β-glycosidic bond at its C1 atom; and fucose is linked to the C3 atom of N-acetylglucosamine through an α-glycosidic bond at its C1 atom.

[0257] "Sialyl Tn antigen" is "Neu5Acα2-3 / 6GalNAc-Ser / Thr", which means that N-acetylneuraminic acid is linked to the C3 or C6 atom of N-acetylgalactosamine via an α-glycosidic bond at its C2 atom, and the N-acetylgalactosamine is linked to serine / threonine via an O-glycosidic bond,

[0258] "Sialyl T antigen" is "Neu5Acα2-3 / 6Galβ1-3GalNAc-Ser / Thr", which means that N-acetylneuraminic acid is linked to the C3 or C6 atom of galactose via an α-glycosidic bond at its C2 atom, the galactose is linked to the C3 atom of N-acetylgalactosamine via a β-glycosidic bond at its C1 atom, and the N-acetylgalactosamine is linked to serine / threonine via an O-glycosidic bond,

[0259] "Louis y ”(Le y ) antigen is "Fucα1-2Galβ1-4(Fucα1-3)GlcNAc", which means that fucose is linked to the C2 atom of galactose via an α-glycosidic bond at its C1 atom, and the galactose is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom; at the same time, a second fucose is linked to the C3 atom of N-acetylglucosamine via an α-glycosidic bond at its C1 atom,

[0260] "Sulfated core 1 glycan" is a sulfated extended form of glycan based on the T antigen.

[0261] A "core 2 glycan" is a glycan based on an extended form of Galβ1-3(GlcNAcβ1-6)GalNAc-Ser / Thr, meaning an extended form of a glycan having galactose linked to the C3 atom of N-acetylgalactosamine via a β-glycosidic bond through the C1 atom of galactose, while N-acetylglucosamine is linked to the C6 atom of N-acetylgalactosamine via a β-glycosidic bond through its C1 atom, said N-acetylgalactosamine being linked to a serine / threonine,

[0262] "Louis a ”(Le a ) antigen is Galβ1-3(Fucα1-4)GlcNAc, which means that galactose is linked to the C3 atom of N-acetylglucosamine through a β-glycosidic bond at its C1 atom; at the same time, fucose is linked to the C4 atom of N-acetylglucosamine through an α-glycosidic bond at its C1 atom.

[0263] “(GlcNAcβ1-4)n” means that N-acetylglucosamine is repeatedly linked to the C4 atom of N-acetylglucosamine through a β-glycosidic bond of its C1 atom,

[0264] "β-D-GlcNAc" means the amide between β-D-glucosamine and acetic acid,

[0265] "GalNAc" means the amide between galactosamine and acetic acid, i.e., N-acetylgalactosamine,

[0266] "Gal-GlcNAc" means galactose linked to N-acetylglucosamine via a non-specific linkage,

[0267] "GlcNAc" means the amide between glucosamine and acetic acid, ie, N-acetylglucosamine.

[0268] "Galα1-3Gal" means that galactose is linked to the C3 atom of galactose via an α-glycosidic bond at its C1 atom,

[0269] "Galβ1-3GalNAc" means that galactose is linked to the C3 atom of N-acetylgalactosamine via a β-glycosidic bond at its C1 atom,

[0270] "α-Gal" means α-galactose,

[0271] "α-GalNAc" means the amide between α-D-galactosamine and acetic acid,

[0272] "(GlcNAc) n " means that N-acetylglucosamine is linked to N-acetylglucosamine via a non-specific linkage,

[0273] "Branched chain (LacNAc) n " is a branched and repeating form of Galβ1,4-GlcNAc, which means a branched and repeating form of galactose linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond at its C1 atom.

[0274] Lectins can be obtained from seeds of leguminous plants, as well as from other plant and animal sources. Lectins may contain binding sites for specific monosaccharides and oligosaccharides (e.g., polysaccharides of glycoproteins). They can agglutinate cells by binding to specific sugar residues in membrane glycoproteins. Preferably, the lectin of the present invention is selected from: Sophora japonica agglutinin II (MAA II) as defined herein; Con A; Dictyosporum aurantium aurantium agglutinin (AAL); Sambucus nigra (SNA-I) lectin; Wisteria floribunda agglutinin (WFL).

[0275] Further preferred lectins of the present invention are shown in the following Table 1. In this context, it is not necessary that the lectins are used in combination with the indicated antibodies or proteins, but this can also be seen as an example of the pairing of the lectins with their recognized glycan structures.

[0276] Particularly preferred lectins according to the invention are those with the following UniProtKB accession numbers (numbering according to v1 numbering in the database): P0DKL3, P02866, P18891, O04366, A0A218PFP3, Q945S3, Q00022, Q6YNX3, Q71QF2, P02872, P18670, Q2UNX8, Q8L5H4, A0A089ZWN7, P05045, P19588, P83410, P17931, P56470, P24146, Q41263, Q39990, Q2 F1K8, G9M5T0, B3XYC5, P02870, P19664, P0DKL3, P49300, A9XX86, Q40423, P16300, P05088, P05087, Q9AVB0, P02867, O24313, Q9SM56, P06750, B9SPG3, Q9BZZ2, P20916, Q9NYZ4, Q96RL6, P05046, P93535, P02876, P10968, P10969, P22972 or P56625 and their corresponding mature forms.

[0277] Exemplary lectins of the invention also include:

[0278] Sophora japonica agglutinin II (MAA II) is a hemagglutinin isolectin from Sophora japonica seeds. It is a sialic acid binding lectin that recognizes oligosaccharides containing terminal sialic acid linked to adjacent galactose residues by α2-3 bonds. It binds the trisaccharide sequence Neu5Acα2-3-Gal-β-1-4-GlcNAc. Preferably, MAA II has SEQ ID NO: 2 (or its mature form).

[0279] Con A is a D-mannose specific lectin originally extracted from the jack bean - Canavalia ensiformis. Preferably, Con A has SEQ ID NO: 3 or SEQ ID NO: 4 (Con A, mature form).

[0280] AAL is a fucose-specific lectin extracted from A. aurantiacus (Auricularia aurantiacus). Preferably, AAL has SEQ ID NO: 5 (or its mature form). Isolation of AAL is described, for example, by (Debray et al., Kochibe et al.).

[0281] Sambucus nigra (SNA-I) lectin is a Neu5Acα2-6)Gal / GalNAc specific lectin extracted from Sambucus nigra (European elder). Preferably, SNA-I has SEQ ID NO: 6 (or its mature form).

[0282] Wisteria floribunda agglutinin (WFL) is a lectin extracted from Wisteria floribunda (Japanese wisteria). Preferably, WFL has SEQ ID NO: 7 (or its mature form).

[0283] Furthermore, suitable lectins within the meaning of the present invention may explicitly include post-translationally processed and mature forms of the lectins disclosed herein.

[0284] As used herein, the term "bead" refers to a small spherical object, for example, made of glass, plastic, metal, agarose, latex, metal nanoparticles or microparticles, metal oxide nanoparticles or microparticles, or a magnetic material. Therefore, preferably, the first bead and / or the further bead are made of glass, plastic, metal, agarose, latex, metal nanoparticles or microparticles, metal oxide nanoparticles or microparticles, or a magnetic material. Preferably, the first bead is a magnetic carrier. Preferably, the further bead is a magnetic carrier. As used herein, the term "magnetic carrier" refers to a particle or bead comprising a magnetic material or substance (e.g., iron or ferritin). Preferably, the magnetic carrier is a magnetic particle or magnetic bead (e.g., a ferritin conjugate). However, for the avoidance of doubt, when referred to herein, the magnetic carrier is not a solid surface, such as a plate, such as an ELISA plate or a microtiter plate.

[0285] As described herein, the one or more additional beads include a label. Preferably, the label is a detectable label. Preferred labels include, but are not limited to, enzymes, radioisotopes, fluorescent proteins, fluorescent dyes, bioluminescent labels or tags (such as biotin). Detectable labels can be any of the various types currently used in the field of in vitro diagnostics, including particulate labels, isotopes, chromophores containing metals such as colloidal gold, including fluorescent markers, biotin, luminescent markers, phosphorescent markers, etc., and enzyme labels that convert a given substrate into a detectable marker, and polynucleotide labels displayed after amplification (e.g., by polymerase chain reaction). Suitable enzyme labels include horseradish peroxidase, polyHRP, alkaline phosphatase, etc., preferably horseradish peroxidase. For example, the marker can be alkaline phosphatase, which can be detected by measuring 1,2-dioxetane substrates such as adamantyl methoxyphosphoryloxyphenyl dioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.13,7}decane}-4-yl)phenyl phosphate disodium (CSPD), and CDP and Or other luminescent substrates well known to those skilled in the art, such as the presence or formation of chemiluminescence after conversion of chelates of suitable lanthanides (such as terbium (III) and europium (III)). The detection means is determined by the selected label. In the case where the label is a microparticle and accumulates at an appropriate level, the appearance of the label or its reaction product can be observed with the naked eye, or with instruments such as spectrophotometers, luminometers, fluorometers, etc., all of which are performed according to standard practice. Therefore, the labeling of the one or more additional beads can be detected based on optical, fluorescent, luminescent, electrochemiluminescent and / or multi-analyte spectrometry (xMAP) readings or means. The labeling of the one or more additional beads can be detected by optical means, such as absorption or visual inspection at a specific wavelength. The labeling of the one or more additional beads can be detected by fluorescent means, such as after excitation at different, usually shorter wavelengths, the emission of a fluorophore at a specific wavelength is determined. The labeling of the one or more additional beads can be detected by electrochemiluminescent means, for example, using the commercially available ELECSYS system of Roche. The label of the one or more additional beads may be detected by multi-analyte profiling (xMAP), for example as described in WO2007 / 075891. As used herein, "tags" may include, but are not limited to, affinity tags that are attached to proteins so that they can be purified from crude biological sources of the proteins using affinity techniques, such as chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag and glutathione-S-transferase (GST), or poly (His) tags, which are widely used protein tags that bind to metal matrices; chromatographic tags used to alter the chromatographic properties of proteins to provide different resolutions in specific separation techniques, such as FLAG-tags; epitope tags, which are short peptide sequences that are chosen because high-affinity antibodies can be reliably produced in many different species, such as ALFA-tags, V5-tags, Myc-tags, HA-tags, Spot-tags, T7-tags and NE-tags; fluorescent tags used to give visual readouts on proteins, such as GFP and its variants; protein tags that can allow specific enzymatic modification (e.g., biotinylation by biotin ligase) or chemical modification (e.g., reaction with FlAsH-EDT2 for fluorescent imaging).

[0286] Preferably, the label is a microperoxidase. As used herein, the term "microperoxidase" or "MP" refers to a heme-containing peptide portion of a cytochrome c (e.g., as shown in SEQ ID NO: 11, cytochrome c from Equus caballus, NCBI reference number: NP_001157486.1) that retains peroxidase activity (e.g., EC 1.11.1.7 enzyme activity, e.g., microperoxidase-11). Preferably, the heme-containing peptide portion of cytochrome c is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NO: 8 (MP-11 peptide), SEQ ID NO: 9 (MP-9 peptide) and SEQ ID NO: 10 (MP-8 peptide). Preferably, the microperoxidase (MP) peptide is selected from the group consisting of SEQ ID NO: 8 (MP-11 peptide), SEQ ID NO: 9 (MP-9 peptide) and SEQ ID NO: 10 (MP-8 peptide).

[0287] Another suitable label is biotin. Therefore, the label is preferably biotin. In this embodiment, biotin can be used as an anchor for adding another label that can bind to biotin. Such another label can be any label described herein in principle, but obviously not biotin itself. Therefore, instead of directly coupling the label to one or more other beads, the (other) label can be "indirectly" coupled to one or more other beads by binding to biotin. Thus, the amplification effect described herein can also be achieved. Preferably, the other label that is or can be bound to the biotin on the one or more other beads includes a biotin binding part, such as streptavidin. It is also envisioned that the label on one or more other beads is a binding partner A (such as biotin), wherein the other label comprises a ligand B (such as streptavidin) that can specifically bind to partner A. "Streptavidin" is a protein purified from the bacterium streptomyces avidinii. Streptavidin homotetramers have a very high affinity for biotin (also known as vitamin B7 or vitamin H). The binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature, with a dissociation constant (K d ) is about 10 -14mol / L. An exemplary amino acid sequence of wild-type streptavidin is: MRKIVVAAIAVSLTTVSITASASADPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ (SEQ ID NO: 1). An exemplary wild-type sequence of streptavidin is also shown in UniProt database entry P22629, version 1 of August 1, 1991. As used herein, streptavidin, for example, as used in the context of the methods or uses described herein, may also encompass streptavidin mutant proteins. Streptavidin mutant proteins are disclosed, for example, in WO2017 / 186669 or WO2014 / 076277. The streptavidin or streptavidin mutein used in the methods and uses of the present invention may be derived from N- or / and C-terminally truncated streptavidin variants. Preferred polypeptides of the present invention comprise the amino acid sequence of minimal streptavidin, the N-terminus of which starts in the region of amino acid positions 10-16 and the C-terminus of which terminates in the region of amino acid positions 133-142. Preferably, such a streptavidin mutein polypeptide corresponds to minimal streptavidin outside the mutation region, the minimal streptavidin comprising an amino acid sequence from position Ala 13 to Ser139, and optionally having an N-terminal methionine residue instead of Ala 13. The numbering of the amino acid positions in the present application refers throughout to the numbering of mature wt-streptavidin (Argarana et al., Nucleic Acids Res. 14 (1986), 1871-1882, see SEQ ID NO: 1), which was also deposited under accession number UniProtKB-P22629, V1 of August 1, 1991. In the context of the methods or uses described herein, "streptavidin" as used herein may also relate to other biotin-binding moieties than streptavidin, such as proteins or aptamers that bind to biotin.

[0288] In one embodiment, wherein the label is biotin and the additional label is bound to the biotin rather than directly to the additional beads, it is possible that the actual detectable label is added to the antibody-protein-lectin complex of step (b) after step (b), for example in optional step (b') or step (c).

[0289] When used herein, "antibody" is a protein comprising one or more polypeptides (comprising one or more binding domains, preferably antigen binding domains) encoded substantially or in part by immunoglobulin genes or fragments of immunoglobulin genes. Preferably, antibodies directed against proteins whose sugar profiles are determined as described herein are not directed against polysaccharides attached to the protein. In other words, antibodies directed against proteins whose sugar profiles are determined as described herein are preferably directed against the protein itself, i.e., against epitopes within the amino acid sequence of the protein. An epitope can be a linear or conformational epitope. It can be a continuous or discontinuous epitope. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as countless immunoglobulin variable region genes. In particular, "antibodies" used herein are tetrameric glycosylated proteins typically composed of two light (L) chains (each about 25 kDa) and two heavy (H) chains (each about 50 kDa). Two light chains, called λ and κ, can be found in antibodies. According to the amino acid sequence of the heavy chain constant domain, immunoglobulins can be divided into five major categories: A, D, E, G and M, and several of these categories can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, wherein IgG is preferred in the context of the present invention. It is also envisioned that an antibody of the present invention has an IgE constant region or a portion thereof that binds to Fcε receptor I. IgM antibodies are composed of 5 basic heterotetrameric units together with an additional polypeptide called J chain, and contain 10 antigen binding sites, while IgA antibodies contain 2-5 basic 4-chain units that can be polymerized to form multivalent aggregates in combination with J chains. In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each light chain includes an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain includes an N-terminal V domain (VH), three or four C domains (CH) and a hinge region. The constant region is not directly involved in the binding of an antibody to an antigen, but may exhibit various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC). If the antibody can exert ADCC, it is preferably of the IgG1 subtype, while the IgG4 subtype does not have the ability to exert ADCC.

[0290] The term "antibody" also includes, but is not limited to, monoclonal, monospecific, poly- or multi-specific antibodies such as bispecific antibodies, humanized, camelized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, transplanted and in vitro generated antibodies, wherein chimeric or humanized antibodies are preferred. The term "humanized antibody" is generally defined as an antibody in which the specific encoding CDRs of the HC and LC have been transferred to an appropriate human variable framework ("CDR transplant"). The term "antibody" also includes scFv, single-chain antibodies, diabodies or tetrabodies, domain antibodies (dAb) and nanobodies. For the purposes of the present invention, the term "antibody" should also include double, three or multimeric antibodies or double, three or multifunctional antibodies with several antigen binding sites. The term also includes antigen binding portions. The term "antibody" also includes FN3 scaffolds, adnectins, affibodies, anticalins, avimers, bicyclic peptides, DARPins, Kunitz domains, Obodies or aptamers such as DNA, RNA or peptide aptamers.

[0291] Preferred antibodies of the present invention include, but are not limited to, anti-PSA, anti-AFP, anti-MUC16, anti-WFDC2, anti-MUC1, anti-ERBB2, anti-CEACAM5, anti-FUT3 or anti-TG antibodies, etc. Further preferred antibodies of the present invention are shown in the following Error! Reference source not found.

[0292] In addition, the term "antibody" as used herein also relates to derivatives of the antibodies (including fragments) described herein. "Derivatives" of antibodies include amino acid sequences that have been altered by the introduction of amino acid residue substitutions, deletions or additions. In addition, derivatives encompass antibodies that have been modified by covalent attachment of any type of molecule to an antibody or protein. Examples of such molecules include sugars, PEG, hydroxyl, ethoxy, carboxyl or amino groups, but are not limited to these. In fact, covalent modifications of the antibodies result in glycosylation, pegylation, acetylation, phosphorylation and amidation, but are not limited to these.

[0293] As used herein, the term "specifically binds" or "directed against" refers to an antibody or fragment or derivative thereof that specifically binds to a target glycoprotein or target polypeptide and does not specifically bind to another protein or polypeptide. The antibodies or fragments or derivatives thereof according to the present invention bind to their respective targets via the variable domains of the antibodies. Generally, when the binding affinity is higher than 10 -6 M, the binding is considered specific. Preferably, the binding affinity is about 10 -11 Up to 10 -8 M(K D ), preferably about 10 -11 Up to 10 -9When the binding affinity is less than 10 μM, the binding is considered specific. If necessary, nonspecific binding can be reduced by changing the binding conditions without substantially affecting specific binding. In the case of glycan binding to lectin, the binding affinity is preferably between 10 -3 Up to 10 -6 (K D ) range. Measuring the corresponding K of glycan binding to lectin D The methods are known in the art and are readily available to those skilled in the art.

[0294] As outlined herein, the antibody is combined with the first bead, and the lectin and the label are coupled to one or more other beads. Chemical methods for coupling antibodies, lectins and labels to beads are well known to those skilled in the art. Antibodies can be, for example, coupled to beads coated with protein A, protein G or protein L, beads coated with a second antibody or beads coated with epoxy resin. Antibodies, labels and lectins can be, for example, coupled to beads coated with streptavidin or fusion tags. Additionally or alternatively, amine coupling chemistry (e.g., using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or N-hydroxysuccinimide (NHS) coupling chemistry) can be used to covalently couple antibodies to beads modified with terminal-COOH groups. Such beads are commercially available, for example, from Thermo Fisher Scientific, Waltham, Massachusetts, USA.

[0295] The present invention also relates to a kit for implementing the method of the present invention for diagnosing whether a subject may be at risk of cancer or may have cancer, the kit comprising antibodies specific to the cancer biomarker protein as defined herein and one or more lectins as defined herein.

[0296] The present invention also relates to a kit for implementing the method of the present invention for diagnosing whether a subject may be at risk of or may have an autoimmune disease, the kit comprising an antibody specific for an autoimmune disease biomarker protein that is IgG and one or more lectins as defined herein.

[0297] The present invention also relates to a kit for implementing the method of the present invention for diagnosing whether a subject may be at risk of or may have an inflammatory disease, the kit comprising antibodies specific to an inflammatory biomarker protein that is IgG, IgA or CRP and one or more lectins as defined herein.

[0298] The present invention also relates to a kit for carrying out a method of diagnosing whether a subject may be at risk of or may have a neurodegenerative disease, the kit comprising antibodies specific for inflammatory disease biomarker proteins, preferably α-synuclein, tau protein or amyloid β protein and isoforms thereof, and one or more lectins as defined herein.

[0299] In another embodiment of the invention, articles and kits containing antibodies or antigen-binding portions thereof that can be used for therapeutic or non-therapeutic applications, such as those described above, are provided. The articles include containers with labels. Suitable containers include, for example, bottles, vials, and test tubes. The container can be made of various materials such as glass or plastic. The container holds a composition including an activating agent, such as described above, which is effective for therapeutic or non-therapeutic applications. The activating agent in the composition is an antibody or antigen-binding portion thereof. The label on the container indicates that the composition is used for a specific treatment or non-therapeutic application, and also indicates instructions for in vivo or in vitro use, such as those described above.

[0300] Kits of the invention typically include a container as described above and one or more additional containers containing materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0301] The present invention is also characterized by the following items:

[0302] 1. A method for determining the sugar profile of a protein, comprising:

[0303] (a) contacting a sample containing the protein with first beads having antibodies against the protein coupled thereto,

[0304] Thus forming an antibody-protein complex,

[0305] (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label and (ii) a lectin,

[0306] thereby forming an antibody-protein-lectin complex; and

[0307] (c) determining the carbohydrate profile of the protein.

[0308] 2. The method according to item 1, further comprising the step (d) of comparing the sugar profile of the protein with a control sugar profile of the protein to determine whether the sugar profile of the protein deviates from the sugar profile of the control sugar profile.

[0309] 3. The method according to any one of the preceding items, further comprising, before step (b) contacting the antibody-glycoprotein complex with one or more additional beads, step (a') enriching the antibody-glycoprotein complex.

[0310] 4. The method according to any one of the preceding items, further comprising a step (b') of enriching the antibody-protein-lectin complex before the step (c) of determining the carbohydrate profile of the protein.

[0311] 5. The method according to any one of the preceding items, wherein the protein is a cancer biomarker protein, an autoimmune disease biomarker protein, an inflammatory disease biomarker protein, or a neurodegenerative disease biomarker protein.

[0312] 6. The method according to item 5, wherein the cancer biomarker protein is an ovarian cancer biomarker protein, a breast cancer biomarker protein, a colorectal cancer biomarker protein, a pancreatic cancer biomarker protein, a prostate cancer biomarker protein, a thyroid cancer biomarker protein, a liver cancer biomarker protein, a lung cancer biomarker protein, a gastric cancer biomarker protein, a testicular cancer biomarker protein or a bladder cancer biomarker protein.

[0313] 7. The method according to item 6, wherein the prostate cancer biomarker protein is β-binding globin, TIMP-1, PSA, fPSA or tPSA.

[0314] 8. The method according to any one of the preceding items, wherein the lectin is specific for the following substances: core fucose, antennal fucose, N-linked oligosaccharides containing Fucα1-6GlcNAc-N-Asn, Fucα1-6 / 3GlcNAc, α-L-Fuc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Gal, Fucα1-6GlcNAc, Manβ1-4GlcNAcβ1-4GlcNAc, branched N-linked hexasaccharides, Manα1-3Man, α-D-Man, (GlcNAcβ1-4) 2-4 ,Galβ1-4GlcNAc, GlcNAcα1-4Galβ1-4GlcNAc, (GlcNAcβ1-4) 2-5, Neu5Ac (sialic acid), Galβ1-3GalNAc-serine / threonine, Galα1-3GalNAc, Galβ1-6Gal, Galβ1-4GlcNAc, Galβ1-3GalNAc, GalN Acα1-3GalNAc, GalNAcα1-3Gal, GalNAcα / β1-3 / 4Gal, α-GalNAc, GalNAcβ1-4Gal, GalNAcα1-3(Fucα1-2)Gal, G alNAcα1-2Gal, GalNAcα1-3GalNAc, GalNAcβ1-3 / 4Gal, GalNAc-Ser / Thr (Tn antigen), Galβ1-3GalNAc-Ser / Thr (T antigen), GalNAcβ1-4GlcNAc (LacdiNAc), α-2,3Neu5Ac (α2-3 linked sialic acid), α-2,6Neu5Ac (α2-6 linked sialic acid), α-2,8Neu5Ac (α2-8 linked sialic acid), sialic acid (α-2,3Neu5Ac, α-2,6Neu5Ac or α-2,8Neu5Ac), Neu5Acα4 / 9-O-Ac-Neu5Ac, Neu5Acα2-3Galβ1-4Glc / GlcNAc, Neu5Acα2-6Gal / GalNAc, N-linked bi-antennary, N-linked tri / tetra-antennary, branched β1-6GlcNAc, Galα1-3(Fucα1-2)Galβ1-3 / 4GlcNAc Ac, Galβ1-3(Fucα1-4)GlcNAc, NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc, Fucα1-2Galβ1-3(Fucα1-4)GlcNAc, Galβ1-4(Fucα1-3)GlcNAc, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, high mannose, sialyl Lewis a (sialylated Le a ) antigen, sialyl Lewis x (sialylated Le x ) Antigen, Lewis x (Le x ) antigen, sialyl Tn antigen, sialyl T antigen, Lewis y (Le y ) antigen, sulfated core 1 glycan, Tn antigen, T antigen, core 2 glycan, Lewis a (Le a ) antigen, (GlcNAcβ1-4) n, β-D-GlcNAc, GalNAc, Gal-GlcNAc, GlcNAc, Galα1-3Gal, Galβ1-3GalNAc, α-Gal, α-GalNAc, (GlcNAc) n , branched chain (LacNAc) n .

[0315] 9. A method for diagnosing whether a subject may be at risk of cancer or may have cancer, comprising

[0316] (a) contacting a sample obtained from the subject containing a cancer biomarker protein with first beads having antibodies against the cancer biomarker protein coupled thereto, thereby forming an antibody-protein complex,

[0317] (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label and (ii) a lectin, thereby forming an antibody-protein-lectin complex; and

[0318] (c) determining the sugar profile of the cancer biomarker protein,

[0319] Wherein a deviation of the sugar profile from a healthy sugar profile of the cancer biomarker protein indicates that the subject may be at risk for cancer or may have cancer.

[0320] 10. A method for diagnosing whether a subject may be at risk of or may have an autoimmune disease, comprising

[0321] (a) contacting a sample obtained from the subject containing an autoimmune disease biomarker protein with first beads having antibodies against the autoimmune disease biomarker protein coupled thereto, thereby forming an antibody-protein complex,

[0322] (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label and (ii) a lectin, thereby forming an antibody-protein-lectin complex; and

[0323] (c) determining the sugar profile of the autoimmune disease biomarker protein,

[0324] Wherein a deviation of the sugar profile from a healthy sugar profile of the autoimmune disease biomarker protein indicates that the subject may be at risk for or may have an autoimmune disease.

[0325] 11. A method for diagnosing whether a subject may be at risk of or may have an inflammatory disease, comprising

[0326] (a) contacting a sample obtained from the subject containing an inflammatory disease biomarker protein with first beads having antibodies against the inflammatory disease biomarker protein coupled thereto, thereby forming an antibody-protein complex,

[0327] (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label and (ii) a lectin, thereby forming an antibody-protein-lectin complex; and

[0328] (c) determining the carbohydrate profile of the inflammatory disease biomarker protein,

[0329] wherein a deviation of the sugar profile from a healthy sugar profile of the inflammatory disease biomarker protein indicates that the subject may be at risk for an inflammatory disease or may have cancer.

[0330] 12. A method for diagnosing whether a subject may be at risk of or may have a neurodegenerative disease, comprising

[0331] (a) contacting a sample obtained from the subject and containing a neurodegenerative disease biomarker protein with first beads having antibodies against the neurodegenerative disease biomarker protein coupled thereto, thereby forming an antibody-protein complex,

[0332] (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label and (ii) a lectin, thereby forming an antibody-protein-lectin complex; and

[0333] (c) determining the sugar profile of the neurodegenerative disease biomarker protein,

[0334] wherein a deviation of the sugar profile from a healthy sugar profile of the neurodegenerative disease biomarker protein indicates that the subject may be at risk for or may have a neurodegenerative disease.

[0335] 13. A kit for implementing the method described in item 9, comprising an antibody specific to a cancer biomarker protein as defined in item 5 and one or more lectins as defined in item 8.

[0336] 14. A kit for implementing the method of item 10, comprising an antibody specific to an autoimmune disease biomarker protein that is IgG and one or more lectins as defined in item 8.

[0337] 15. A kit for implementing the method of item 11, comprising an antibody specific to an inflammatory biomarker protein that is IgG, IgA or CRP and one or more lectins as defined in item 8.

[0338] 16. A kit for implementing the method of item 12, comprising antibodies specific to neurodegenerative biomarker proteins, preferably α-synuclein, tau protein or amyloid β protein and isoforms thereof, and one or more lectins as defined in item 8.

[0339] 17. The method according to any of the preceding items, wherein the first beads and the further beads are contacted with the sample simultaneously.

[0340] 18. The method according to any of the preceding items, wherein the further beads are contacted with the sample immediately after contacting the first beads with the sample.

[0341] 19. The method according to any one of the preceding items, wherein the first beads are contacted with the sample immediately after the second beads are contacted with the sample.

[0342] 20. The method according to any of the preceding items, wherein during performance of the method according to any of the preceding items, the first bead and the further beads are in solution.

[0343] 21. The method according to any of the preceding items, wherein the first bead and / or the further beads are made of glass, plastic, metal, agarose, latex, metal nanoparticles or microparticles, metal oxide nanoparticles or microparticles or magnetic material.

[0344] 22. The method according to any of the preceding items, wherein the label of the additional beads is an enzyme, a radioisotope, a fluorescent protein, a fluorescent dye, a bioluminescent marker or a tag (such as biotin).

[0345] 23. The method according to any of the preceding items, wherein the labeling of the additional beads is detected based on optical, fluorescent, luminescent, electrochemiluminescent and / or multi-analyte profiling (xMAP) readouts.

[0346] 24. The method according to any of the preceding items, wherein for each of the one or more additional beads for each carbohydrate detected by lectin, a different label is used in combination.

[0347] ****

[0348] It should be noted that, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" used herein include plural references. Thus, for example, reference to "an agent" includes one or more such different agents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that can modify or replace the methods described herein.

[0349] Unless otherwise indicated, the term "at least" before a series of elements will be understood to refer to each element in the series. Those skilled in the art will recognize or use routine experimentation to determine many equivalents of the specific embodiments of the invention described herein. The present invention is intended to cover such equivalents.

[0350] The term "and / or" wherever used herein includes the meanings of "and," "or," and "all or any other combination of the elements connected by the term."

[0351] The terms "less than" or "greater than" do not include specific numbers.

[0352] For example, less than 20 means less than the indicated number. Similarly, greater than or above means greater than or above the indicated number, for example greater than 80% means greater than or above 80% of the indicated number.

[0353] In this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of the stated integers or steps or sets of integers or steps, but not the exclusion of any other integers or steps or sets of integers or steps. When used herein, the term "comprising" can be replaced with the term "containing" or "including" or, as is sometimes used herein, with the term "having". When used herein, "consisting of excludes any element, step or ingredient not specified.

[0354] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0355] The term "about" or "approximately" or "substantially" as used herein means within 20%, preferably within 15%, preferably within 10%, and more preferably within 5% of a given value or range. It also includes specific numbers, such as "about 20" includes the number 20.

[0356] It should be understood that the present invention is not limited to the specific methodology, protocols, materials, reagents and substances, etc. described herein, as these can vary. The terminology used herein is only used for the purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which is limited only by the claims.

[0357] All publications cited in this specification (including all patents, patent applications, scientific publications, guides, etc.) are incorporated herein by reference in their entirety, whether before or after. Nothing herein shall be construed as an admission that the present invention is not earlier than such publications due to prior invention. In the event that the material incorporated by reference is inconsistent or inconsistent with this specification, this specification shall supersede any such material.

[0358] The contents of all literature and patent documents cited herein are incorporated by reference in their entirety.

[0359] Example

[0360] The present invention and its advantages will be better understood from the following examples which are given for illustrative purposes only. These examples do not limit the scope of the present invention in any way.

[0361] Example 1

[0362] In this example, the use of two types of beads in cancer diagnostics based on glycoprofiling of proteins by glycan analysis (see Figure 1 ). First, magnetic beads ("first beads", or beads 1) are coupled to antibodies against the free form of PSA (fPSA) using standard bioconjugation protocols. The anti-fPSA antibody is selected in a manner that binds to an epitope on fPSA that is away from the glycan epitope (Asn61) occupied by N-glycans on fPSA. Another type of beads (non-magnetic beads; i.e., "one or more additional beads", or beads 2) is coupled to a lectin that is capable of selectively recognizing cancer-specific glycans using a standard bioconjugation protocol. The lectin is co-immobilized with a peroxidase, any other enzyme, or any label that is capable of producing an optical, fluorescent, chemiluminescent, and electrochemiluminescent signal. Alternatively, an enzyme or label is attached to a bead 2 that has been modified with a lectin. In one approach, the inventors produced beads 2 modified with different enzymes and labels, allowing us to perform analysis in a parallel / multiplex / array format.

[0363] The beads 1 coupled with anti-fPSA antibodies are incubated with samples containing fPSA to react for a considerable time, the complex is separated by magnetic force, and the complex attached to the magnetic beads is washed with washing buffer. In the next step, the complex is incubated with beads 2 patterned by lectin and label (enzyme or any other label producing signal), and the whole complex is separated by magnetic force, and washed by washing buffer. In ELISA plates, in any test tube, in solution phase or in the flow system of a highly automated machine, the assay is carried out. The assay can be repeated with one or more other lectins. In this form, during the analysis for diagnosing prostate cancer, the assay shows increased sensitivity and specificity and low background signals. In addition, a low detection limit is confirmed. The method is applicable to the sugar spectrum analysis of any other glycoprotein produced by prostate and any other organ, and is therefore applicable to the diagnosis of other cancer types.

[0364] Example 2

[0365] Figure 2 The assay used two types of nanoparticles, namely integrated gold nanoparticles with a size of 40nm and magnetic particles with a size of 130nm. In this case, only fPSA antibodies were bound to the surface of 130nm magnetic particles (MNPs). Horseradish peroxidase (HRP) and lectin were bound to the surface of gold nanoparticles with a size of 40nm, creating a sandwich configuration in the presence of the analyte, namely fPSA protein. The sandwich method was then used to analyze 110 real human serum samples. The results showed that fPSA sugar profiling can provide significantly better clinical parameters compared to tPSA analysis (total PSA, a prostate cancer screening biomarker). ROC (Receiver Operating Curve) curve showing the difference between tPSA (black line) and PGI+ index (light gray line) ( Figure 2 ) provided the following clinical parameters: the AUC for tPSA was 0.686 (CI95% = [0.581, 0.781], specificity = 0.654, sensitivity = 0.690, accuracy = 0.673), and the AUC for PGI+ (glycogen profile analysis fPSA) was 0.803 (CI95% = [0.707, 0.890], specificity = 0.712, sensitivity = 0.828, accuracy = 0.773).

Claims

1. A method for determining the sugar profile of a protein, comprising: (a) contacting a sample containing the protein with first beads having antibodies against the protein coupled thereto, Thus forming an antibody-protein complex, (b) contacting the antibody-protein complex with one or more additional beads, each additional bead having coupled thereto (i) a label that amplifies the signal generated and (ii) a lectin, thereby forming an antibody-protein-lectin complex; and (c) determining the carbohydrate profile of the protein.

2. The method according to claim 1, further comprising the step (d) comparing the sugar profile of the protein with a reference sugar profile of the protein to determine whether the sugar profile of the protein deviates from the sugar profile of the reference sugar profile.

3. The method according to any one of the preceding claims, further comprising step (a') enriches the antibody-glycoprotein complexes prior to step (b) contacting the antibody-glycoprotein complexes with one or more additional beads; and / or Prior to step (c) determining the carbohydrate profile of the protein, step (b') enriches the antibody-protein-lectin complex.

4. The method according to any one of the preceding claims, wherein the protein is a cancer biomarker protein, an autoimmune disease biomarker protein, an inflammatory disease biomarker protein, or a neurodegenerative disease biomarker protein.

5. The method according to any one of the preceding claims, wherein the lectin is specific for core fucose, antennal fucose, N-linked oligosaccharides containing Fucα1-6GlcNAc-N-Asn, Fucα1-6 / 3GlcNAc, α-L-Fuc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Gal, Fucα1-6GlcNAc, Manβ1-4GlcNAcβ1-4GlcNAc, branched N-linked hexasaccharides, Manα1-3Man, α-D-Man, (GlcNAcβ1-4) 2-4 ,Galβ1-4GlcNAc, GlcNAcα1-4Galβ1-4GlcNAc, (GlcNAcβ1-4) 2-5 , Neu5Ac (sialic acid), Galβ1-3GalNAc-serine / threonine, Galα1-3GalNAc, Galβ1-6Gal, Galβ1-4GlcNAc, Galβ1-3GalNAc, GalN Acα1-3GalNAc, GalNAcα1-3Gal, GalNAcα / β1-3 / 4Gal, α-GalNAc, GalNAcβ1-4Gal, GalNAcα1-3(Fucα1-2)Gal, G alNAcα1-2Gal, GalNAcα1-3GalNAc, GalNAcβ1-3 / 4Gal, GalNAc-Ser / Thr (Tn antigen), Galβ1-3GalNAc-Ser / Thr (T antigen), GalNAcβ1-4GlcNAc (LacdiNAc), α-2,3Neu5Ac (α2-3 linked sialic acid), α-2,6Neu5Ac (α2-6 linked sialic acid), α-2,8Neu5Ac (α2-8 linked sialic acid), sialic acid (α-2,3Neu5Ac, α-2,6Neu5Ac or α-2,8Neu5Ac), Neu5Acα4 / 9-O-Ac-Neu5Ac, Neu5Acα2-3Galβ1-4Glc / GlcNAc, Neu5Acα2-6Gal / GalNAc, N-linked bi-antennary, N-linked tri / tetra-antennary, branched β1-6GlcNAc, Galα1-3(Fucα1-2)Galβ1-3 / 4GlcNAc Ac, Galβ1-3(Fucα1-4)GlcNAc, NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc, Fucα1-2Galβ1-3(Fucα1-4)GlcNAc, Galβ1-4(Fucα1-3)GlcNAc, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, high mannose, sialyl Lewis a (sialylated Le a ) antigen, sialyl Lewis x (sialylated Le x ) Antigen, Lewis x (Le x ) antigen, sialyl Tn antigen, sialyl T antigen, Lewis y (Le y ) antigen, sulfated core 1 glycan, Tn antigen, T antigen, core 2 glycan, Lewis a (Le a ) antigen, (GlcNAcβ1-4) n , β-D-GlcNAc, GalNAc, Gal-GlcNAc, GlcNAc, Galα1-3Gal, Galβ1-3GalNAc, α-Gal, α-GalNAc, (GlcNAc) n , branched (LacNAc) n .

6. The method of claim 4, wherein the protein is a cancer biomarker protein, and wherein a deviation of the sugar profile from a healthy sugar profile of the cancer biomarker protein indicates that the subject may be at risk for cancer or may have cancer.

7. The method of claim 6, wherein the cancer biomarker protein is any one of an ovarian cancer biomarker protein, a breast cancer biomarker protein, a colorectal cancer biomarker protein, a pancreatic cancer biomarker protein, a prostate cancer biomarker protein, a thyroid cancer biomarker protein, a liver cancer biomarker protein, a lung cancer biomarker protein, a gastric cancer biomarker protein, a testicular cancer biomarker protein, or a bladder cancer biomarker protein.

8. The method of claim 7, wherein the prostate cancer biomarker protein is any one of β-haptoglobin, TIMP-1, PSA, fPSA or tPSA.

9. The method of claim 4, wherein the protein is an autoimmune disease biomarker protein, and wherein a deviation of the sugar profile from a healthy sugar profile of the autoimmune disease biomarker protein indicates that the subject may be at risk for or may have an autoimmune disease.

10. The method of claim 4, wherein the protein is an inflammatory disease biomarker protein, and wherein a deviation of the sugar profile from a healthy sugar profile of the inflammatory disease biomarker protein indicates that the subject may be at risk for or may have an inflammatory disease.

11. The method of claim 4, wherein the protein is a neurodegenerative disease biomarker protein, and wherein a deviation of the sugar profile from a healthy sugar profile of the neurodegenerative disease biomarker protein indicates that the subject may be at risk for or may have a neurodegenerative disease.

12. A kit for implementing (a) The method of claim 6, wherein the kit comprises an antibody specific for the cancer biomarker protein as defined in claim 7 and one or more lectins as defined in claim 5; (b) The method of claim 9, wherein the kit comprises an antibody specific for the autoimmune disease biomarker protein that is IgG and one or more lectins as defined in claim 5; (c) The method of claim 10, wherein the kit comprises an antibody specific for the inflammatory biomarker protein that is IgG, IgA or CRP and one or more lectins as defined in claim 5; or (d) The method of claim 11, wherein the kit comprises an antibody specific for the neurodegenerative biomarker protein and one or more lectins as defined in claim 5, wherein the neurodegenerative biomarker protein is α-synuclein, tau protein or amyloid β protein and its isoforms.

13. The method according to any one of claims 1 to 11, (a) wherein the first beads and the further beads are contacted with the sample simultaneously; (b) wherein said further beads are contacted with said sample immediately after contacting said first beads with said sample; or (c) wherein the first beads are contacted with the sample immediately after the second beads are contacted with the sample.

14. The method of any one of claims 1-11 and 13, wherein the first bead and the further beads are in solution during performance of the method of any one of the preceding claims.

15. The method according to any one of claims 1-11, 13 and 14, wherein the first beads and / or the further beads are made of glass, plastic, metal, agarose, latex, metal nanoparticles or microparticles, metal oxide nanoparticles or microparticles or magnetic material.

16. The method according to any one of claims 1 to 11 and 13 to 15, (a) wherein the label of the additional beads is an enzyme, a radioisotope, a fluorescent protein, a fluorescent dye, a bioluminescent marker or a tag (such as biotin); and / or (b) wherein the label of the additional beads is detected based on an optical, fluorescent, luminescent, electrochemiluminescent and / or multi-analyte profiling (xMAP) readout.

17. The method of any one of claims 1-11 and 13-16, wherein for each of the one or more additional beads for each carbohydrate detected by lectin, a different label is used in combination.

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