Means and methods for high throughput glycoprofiling of proteins

Abstract

The present invention discloses a method for determining the glycoprofile of a protein, comprising the steps of: (a) contacting a sample containing the protein with a first bead to which an antibody against the protein is coupled, to form an antibody-protein complex; (b) contacting the antibody-protein complex with one or more additional beads, each of which is coupled with (i) a label that amplifies the signal generated and (ii) a lectin, to form an antibody-protein-lectin complex; and (c) determining the glycoprofile of the protein.Furthermore, methods for diagnosing cancer, autoimmune disease, and inflammatory disease, as well as kits for carrying out the methods disclosed herein, are disclosed. TIFF2025512055000036.tif75170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to European Patent Application No. 22167920.2, filed April 12, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] TECHNICAL FIELD OF THEINVENTION The present invention provides a method for determining the glycoprofile of a protein, comprising the steps of: (a) contacting a sample containing the protein with first beads to which an antibody against the protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) a label that amplifies the signal generated; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the protein Additionally disclosed are methods for diagnosing cancer, autoimmune diseases, and inflammatory diseases, as well as kits for carrying out the methods disclosed herein. [Background technology]

[0003] background Glycans are present on a variety of different proteins, where they affect the transport, stability, and folding of the protein, ultimately altering its biochemical and biophysical properties. In addition, glycans can mediate proteolytic patterns or directly mediate both ligand-receptor interactions, oncogenic signaling, immune recognition, migration, and cell-cell and cell-matrix adhesion. Thus, certain glycans may exert a selective advantage on tumor cells. Thus, the presence of certain glycans, or the presence of certain glycans on certain proteins, can be used as biomarkers, for example, for the diagnosis of cancer.

[0004] 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 sugars that are part of other molecules. These binding molecules can be used to analyze the presence or absence of specific glycan structures in assays such as enzyme-linked immunosorbent assays (ELISA), enzyme-linked lectin assays (ELLA), magnetic ELLA (MELLA), or parallel / multiplex Luminex-like assays that use optical, fluorescent, luminescent, or electrochemiluminescent readouts.

[0005] WO2019 / 185515 (Patent Document 1) discloses a method for determining the glycoprofile of a protein of interest, which includes enriching the protein of interest with an antibody, followed by contacting the complex thus created with one or more lectins. Li et al. (2013), Clinical Chemistry, 59(1):315-324 (Non-Patent Document 1) also discloses a method for determining the glycoprofile of a protein, in which the protein of interest is enriched via an antibody coupled to a bead, followed by contacting with a labeled lectin.

[0006] Carlstrom et al. (2018), Technical note. AlphaLISA Technology (Non-Patent Document 2) discloses a method for determining the glycoprofile of an antibody, in which the antibody is bound to a lectin, itself coupled to a bead (donor bead), and further to a protein G, coupled as a modification to an AlphaLISA acceptor bead. Thus, each bead is conjugated to said antibody, and if the glycan binds to the lectin of the donor bead, detection of the glycosylation state of the antibody is thereby possible. If there is an interaction, the donor bead approaches the acceptor bead, and excitation of the donor bead results in a luminescence signal from the acceptor bead (protein G AlphaLISA bead).

[0007] Additionally, Chen Li et al. (2011), Electrophoresis, 32(15): 2028-2035 (Non-Patent Document 3) disclose a method for profiling the glycosylation pattern of proteins using a protein-specific capture antibody coupled to beads. In such a sandwich assay, only one bead is applied and a fluorescently labeled lectin is used as a detection reagent. The same applies mutatis mutandis to Jun Natsuki et al. (2005), Biotechnology and Bioengineering, 93(2): 225-230 (Non-Patent Document 4). In both prior art documents commenting on the antibody-lectin assay as a fairly reliable tool for glycosylation profiling, there is no motivation or incentive to further amend such antibody-lectin assay. That is because, for example, Chen Li et al. (2011) clearly emphasize that future studies should change the whole assay to multiplex with additional antibodies on beads instead of using a second bead for detection.

[0008] However, there remains a continuing need for further improved methods for determining the glycoprofile of proteins. In particular, these methods should preferably have lower detection limits, higher detection sensitivity and selectivity, especially for cancer-specific markers, with shorter analysis times. The present invention aims to address this need. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2019 / 185515 [Non-patent literature]

[0010] [Non-Patent Document 1] Li et al. (2013), Clinical Chemistry, 59(1):315-324 [Non-Patent Document 2] Carlstrom et al. (2018), Technical note. AlphaLISA Technology [Non-Patent Document 3] Chen Li et al. (2011), Electrophoresis, 32(15): 2028-2035 [Non-Patent Document 4] Jun Natsuki et al. (2005), Biotechnology and Bioengineering, 93(2): 225-230 Summary of the Invention

[0011] This need is solved by the subject matter as defined in the claims and in the embodiments described herein.

[0012] Thus, the present invention provides a method for determining the glycoprofile of a protein, comprising the steps of: (a) contacting a sample containing the protein with first beads to which an antibody against the protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) a label that amplifies the signal generated; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the protein The present invention relates to a method comprising the steps of:

[0013] The method of the present invention may further comprise a step (d) of comparing the glycoprofile of the protein with a control glycoprofile of the protein to determine whether the glycoprofile of the protein may deviate from the glycoprofile of the control glycoprofile of the protein.

[0014] The method of the invention may further comprise a step (a') of concentrating the antibody-glycoprotein complexes prior to step (b) of contacting said antibody-glycoprotein complexes with one or more additional beads.

[0015] The method of the invention may further comprise a step (b') of concentrating said antibody-protein-lectin complexes prior to step (c) of determining the glycoprofile of said protein.

[0016] 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 colon 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-haptoglobin, TIMP-1, PSA, fPSA, or tPSA. Preferably, the protein is a neurodegenerative disease biomarker protein, more preferably an α-synuclein, a tau protein, or an amyloid β protein and its isoforms.

[0017] Preferably, the lectin is selected from the group consisting of core fucose, antennary fucose, Fucα1-6GlcNAc-N-Asn-containing N-linked oligosaccharides, 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, GalNAcα1-3GalNAc, GalNAcα1-3Gal, GalNAcα / β1-3 / 4Gal, α-GalNAc, GalNAcβ1-4Gal, GalNAcα1-3(Fucα1-2)Gal, Gal NAcα1-2Gal, 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), 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 biantennary, N-linked tri / tetraantennary, branched β1-6GlcNAc, Galα1-3(Fucα1-2)Galβ1-3 / 4G lcNAc, 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 (Sialyl Le a ) antigen, sialyl Lewis x (Sialyl 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 , branching (LacNAc) n It is specific to

[0018] The invention may further include the method as defined elsewhere herein, wherein the protein is a cancer biomarker protein and deviation of the glycoprofile from a healthy glycoprofile of the cancer biomarker protein indicates that the subject may be at risk of or may be afflicted with cancer.

[0019] The invention may further include the method as defined elsewhere herein, wherein the protein is an autoimmune disease biomarker protein and deviation of the glycoprofile from a healthy glycoprofile of the autoimmune disease biomarker protein indicates that the subject may be at risk for or may be suffering from an autoimmune disease.

[0020] The invention may further include the method as defined elsewhere herein, wherein the protein is an inflammatory disease biomarker protein and deviation of the glycoprofile from a healthy glycoprofile of the inflammatory disease biomarker protein indicates that the subject may be at risk for or may be suffering from an inflammatory disease.

[0021] The invention may further include the method as defined elsewhere herein, wherein the protein is a neurodegenerative disease biomarker protein and deviation of the glycoprofile from a healthy glycoprofile of the neurodegenerative disease biomarker protein indicates that the subject may be at risk of or may be suffering from a neurodegenerative disease.

[0022] The present invention further relates to a kit for performing a method of diagnosing whether a subject may be at risk for or suffer from a cancer of the present invention, comprising an antibody specific for a cancer biomarker protein as defined herein and one or more lectins as defined herein.

[0023] The present invention further relates to a kit for performing a method of diagnosing whether a subject may be at risk for or suffer from an autoimmune disease of the present invention, comprising an antibody specific for an autoimmune disease biomarker protein, which is IgG, and one or more lectins as defined herein.

[0024] The present invention further relates to a kit for carrying out a method of diagnosing whether a subject may be at risk for or suffer from an inflammatory disease of the present invention, comprising an antibody specific for an inflammatory biomarker protein, which is IgG, IgA, or CRP, and one or more lectins as defined herein.

[0025] The present invention further relates to a kit for performing a method of diagnosing whether a subject may be at risk for or suffer from a neurodegenerative disease, comprising an antibody specific for a neurodegenerative disease biomarker protein that is alpha-synuclein, tau protein, or amyloid beta protein and its isoforms, and one or more lectins as defined herein.

[0026] Preferably, said first bead and said further beads are contacted with said sample simultaneously.

[0027] Preferably, said further beads are contacted with said sample immediately after said first beads are contacted with said sample.

[0028] Preferably, the first beads are contacted with the sample immediately after the second beads are contacted with the sample.

[0029] Preferably, said first beads and said further beads are in solution during carrying out the method according to any one of the preceding claims.

[0030] Preferably, said first bead and / or said further bead are made of glass, plastic, metal, agarose, latex, metal nanoparticles or microparticles, metal oxide nanoparticles or microparticles, or a magnetic material.

[0031] Preferably, the label of said further bead is an enzyme, a radioisotope, a fluorescent protein, a fluorescent dye, a bioluminescent label, or a tag (eg biotin).

[0032] The label of said one or more additional beads may be detected based on optical, fluorescent, luminescent, electrochemiluminescent, and / or multi-analyte profiling (xMAP) readouts or means.

[0033] Preferably, a different label is used in combination for each of one or more additional beads for each carbohydrate detected by the lectin. [Brief description of the drawings]

[0034] The invention will be better understood with reference to the detailed description, taken in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0035] [Figure 1] 1 illustrates an exemplary scheme of an exemplary embodiment of the present invention. A first bead (B1), a further bead (B2) and the protein (analyte) are illustrated. [Diagram 2] Illustrated is a depiction of the assay setup for high-throughput fPSA glycoprofiling (without any washing steps). Anti-fPSA modified magnetic nanoparticles (1) are mixed with the sample containing the analyte, i.e. fPSA (2), in a first chamber and then held at the bottom of a second chamber by a magnetic field. Through this second chamber, lectin / peroxidase modified nanoparticles are first introduced (3) and then washed away by a substrate solution (4). After a short period of time, the solution is pumped into the spectrophotometer chamber (5) and a color change, which is the signal, is detected. Inset: ROC curve showing the difference between tPSA (black line) and PGI+ index (light grey line) in this configuration, with AUC values ​​of 0.686 (CI95%=[0.581;0.781], specificity=0.654, sensitivity=0.690, accuracy=0.673) for tPSA and 0.803 (CI95%=[0.707;0.890], specificity=0.712, sensitivity=0.828, accuracy=0.773) for PGI+, respectively. The amount of samples used in the study was 110 in total (58 BPH patients and 52 PCa patients). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description of the Invention The present invention is described in detail below and further illustrated by the accompanying examples and figures.

[0037] As outlined above, the present invention describes a method for determining the glycoprofile of a (protein of interest). This method can be described in an exemplary embodiment as follows (see also Figures 1 and 2, or Examples 1 or 2). First, a sample containing a protein (of interest, "analyte" in Figure 1; fPSA in Figure 2) is contacted with a first bead ("B1" in Figure 1; "MNPs+Ab" in Figure 2) to which an antibody against the protein is coupled. Thus, the protein is captured on the first bead. The protein may or may not be concentrated, 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 contacted with one or more additional beads ("B2" in Figure 1; nanoparticles containing HRP and lectin in Figure 2). These additional beads contain a lectin that specifically binds to the glycan of the protein, and a label. Thus, the further bead only binds to the first bead-antibody-protein complex if the protein carries a glycan that the lectin specifically binds to, or in other words, has a glycoprofile that is detected by the lectin. Thus, even if only one protein with the glycan structure of interest / to which the lectin binds is bound to the first bead, the lectin on one or more further beads, which itself contains a label, ideally multiple labels, can bind. All these labels on the further beads are active and provide a signal, or a processed signal, even if only one lectin binds. This can be explained as an amplification effect. Thus, as shown in the examples, the combination of lectin and label on a single (further / second) bead allows very low levels of detection. An additional advantage is a higher sensitivity and selectivity of detection, especially for cancer-specific biomarkers. Additionally, the time required for analysis is reduced.In Carlstrom et al. (2018), whose sandwich assay is also clearly distinguished from the method of the present invention in which an antibody against a glycoprotein is used instead of protein G, there is no such amplification effect according to the present invention due to the label coupled to the further / second bead which also contains a lectin as described above. In Carlstrom et al. (2018), the detectable signal is only generated by the acceptor bead coupled to protein G (such beads also do not contain any lectin).

[0038] Both WO 2019 / 185515 and Li et al. (2013), Clinical Chemistry, 59(1):315-324 disclose methods for determining glycoprofiles, but neither describes one or more additional beads, each coupled with (i) a label and (ii) a lectin.

[0039] Thus, the present invention provides a method for determining the glycoprofile of a protein, comprising the steps of: (a) contacting a sample containing the protein with first beads to which an antibody against the protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) Signs; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the protein The present invention relates to a method comprising the steps of:

[0040] The term "glycoprofile of a protein" refers to the carbohydrate structure of a protein (of interest), e.g., the composition and / or structure of covalently attached carbohydrates, e.g., the amount, presence, or absence of covalently attached carbohydrates. The term "glycoprofiling" or "determining a glycoprofile" refers to determining the carbohydrate structure (e.g., the composition and / or structure of covalently attached carbohydrates, e.g., the amount, presence, or absence of covalently attached carbohydrates) on said protein (of interest).

[0041] The method of the present invention can be used to determine whether a protein has a specific glycoprofile, or in other words, whether it carries a specific glycan. It can also be used to distinguish whether a protein carries a glycan that indicates a disease. Thus, a protein with a control glycoprofile, i.e., a protein with a known glycoprofile, can be used as a standard or positive control, and can be compared with the signal obtained by the protein present in, for example, a sample (obtained from a subject). If there is a deviation, this can indicate another glycoprofile. Thus, the method of determining the glycoprofile of a protein of the present invention can further include a step (d) of comparing the glycoprofile of the protein with the control glycoprofile of the protein to determine whether the glycoprofile of the protein may deviate from the glycoprofile of the control glycoprofile of the protein.

[0042] However, the present invention is not limited to the detection of 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, "glycoprofiling" includes the determination of more than one, e.g., two, three, four, five, or more than five different carbohydrate structures (e.g., the composition and / or structure of the covalently attached carbohydrates, e.g., the amount, presence, or absence of the covalently attached carbohydrates) on the protein of interest. Advantageously, a different label is used for each different glycan structure analyzed. Thereby, a specific pair of one specific lectin and one specific label coupled to one specific additional bead is provided. Also, different detection modes may be combined, for example based on fluorescent, luminescent, or chemiluminescent labels that provide a corresponding signal. Thus, preferably, different labels are used in combination for each of one or more additional beads for each carbohydrate detected by the lectin.

[0043] In the method of the present invention, the first bead and / or one or more additional beads can be concentrated. This can be used to further reduce background signals. As used herein, "concentrating" describes the process of increasing the amount of beads / complexes / substances in a mixture. Thus, the method of determining the glycoprofile of a protein of the present invention can further comprise a step (a') of concentrating the antibody-glycoprotein complex before step (b) of contacting the antibody-glycoprotein complex with one or more additional beads. Furthermore, the method of determining the glycoprofile of a protein of the present invention can further comprise a step (b') of concentrating the antibody-protein-lectin complex before step (c) of determining the glycoprofile of the protein. For the sake of clarity, and merely to state the obvious, said steps (a') and / or (b') may also be added in a method for diagnosing whether a subject may be at risk for or may be affected by cancer, for diagnosing whether a subject may be at risk for or may be affected by an autoimmune disease, or for diagnosing whether a subject may be at risk for or may be affected by an inflammatory disease of the invention, or for diagnosing whether a subject may be at risk for or may be affected by a neurodegenerative disease.

[0044] Due to the robustness of the present invention, it is possible that the first bead and the further bead can be contacted with the sample simultaneously or immediately in succession. Thus, the first bead and the further bead can be contacted with the sample simultaneously. Alternatively, the first bead can be contacted with the sample immediately after the further bead is contacted with the sample. Alternatively, the first bead can 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) is not necessarily set by the designation of steps (a) and (b). However, it is preferred that step (a) is followed by step (b) which is followed by step (c).

[0045] Additionally, the first bead and the further bead are preferably in solution during the method described herein.In this context, "in solution" means that the first bead and the further bead are not particularly held by magnetic force or coupled to a solid material such as a microplate, a column or a reaction tube.In one embodiment, the first bead is not held by magnetic force or coupled to a solid material such as a microplate, a column or a reaction tube.In one embodiment, the one or more further beads are not held by magnetic force or coupled to a solid material such as a microplate, a column or a reaction tube.

[0046] The protein (to be glycoprofiled by the method of the present invention) is not particularly limited. However, the protein is preferably a glycoprotein. The protein of interest is preferably a protein whose glycoprofile is associated with a disease, since the presence or absence of a particular glycan structure on a protein may be important for the diagnosis or prognosis of the disease. The term "glycoprotein" (or "glycosylated protein") as used herein means a protein containing one or more N-, O-, S-, or C-covalently linked carbohydrates of various types, ranging from monosaccharides to branched oligosaccharides or polysaccharides (including modifications thereof, such as the addition of sulfo or phospho groups). N-linked glycans are carbohydrates attached to the -NH2 group of asparagine. O-linked glycans are carbohydrates attached to the -OH group of serine, threonine, or hydroxylated amino acids. S-linked glycans are carbohydrates attached to the -SH group of cysteine. C-linked glycans are carbohydrates attached to tryptophan via a CC bond.

[0047] The term "carbohydrate" has the stoichiometric formula C n (H2O) nThe general term "carbohydrate" refers to compounds having the formula: (e.g., aldoses and ketoses). The general term "carbohydrate" includes monosaccharides, oligosaccharides, and polysaccharides, as well as substances derived from monosaccharides by reduction of the carbonyl group (alditols), by oxidation of one or more terminal groups to carboxylic acids, or by replacement of one or more hydroxyl groups by hydrogen atoms, amino groups, thiol groups, or similar groups. Also included are derivatives of these compounds.

[0048] As already described herein, the presence of a particular glycoprofile or glycan on a protein of interest may be associated with the diagnosis of a particular disease, such as cancer, an autoimmune disease, an inflammatory disease, or a neurodegenerative disease. Some combinations of proteins (of interest, i.e. biomarker proteins) and glycan structures (A) are known to be indicative of disease. Specific combinations of disease-indicating (proteins of interest) and glycans, as well as antibodies and lectins that bind to particular glycan structures, are exemplified in Fehler! Verweisquelle konnte nicht gefunden werden. Thus, the methods and uses of the present invention can be used in the diagnosis of diseases, such as cancer, an autoimmune disease, an inflammatory disease, or a neurodegenerative disease. Thus, the presence of said particular glycoprofile or glycan structure may be indicative of diseases, such as cancer, an autoimmune disease, an inflammatory disease, or a neurodegenerative disease.

[0049] In this 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.

[0050] As used herein, "autoimmune disease" refers to a group of diseases characterized by the disease associated with the production of antibodies against one's own tissue.Non-limiting examples of autoimmune disease 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 postviral encephalomyelitis, Addison's disease, autoimmune enteropathy, primary biliary cirrhosis, Goodpasture's syndrome, Hashimoto's thyroiditis, myasthenia gravis, myxedema, pemphigoid, rheumatoid arthritis, Sjogren's syndrome, sympathetic ophthalmia, both types of lupus erythematosus, thyrotoxicosis, ulcerative colitis, multiple sclerosis, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, and psoriasis.

[0051] As used herein, "inflammatory disease" refers to a group of diseases characterized by the disorder and / or malfunction of the body's inflammatory mechanism.Non-limiting examples of inflammatory disease 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 suppurativa, hypersensitivity, 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.

[0052] As used herein, "neurodegenerative disease" refers to a group of diseases characterized by brain damage and / or abnormal function. Non-limiting examples of inflammatory diseases include Parkinson's disease, Alzheimer's disease, and other forms of tauopathy, primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia and parkinsonism linked to chromosome 17, vacuolar tauopathy, Ritiko-Bodig disease, ganglioglioma and gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, pantothenate kinase-associated neurodegeneration, and lipofuscinosis.

[0053] As used herein, "cancer" refers to a group of diseases characterized by uncontrolled proliferation of abnormal cells in the body. Unregulated cell division can result in the formation of malignant tumors, or cells that can invade adjacent tissues and 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, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colon cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer (or carcinoma), gastric cancer. cancer), germ cell tumors, childhood sarcomas, sinonasal natural killer, melanoma (e.g., metastatic melanoma such as cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcomas, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, viruses and hematological malignancies derived from either of the two major blood cell lineages, i.e., myeloid cell lineage (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lineage (producing B, T, NK, and plasma cells), e.g., all types of leukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic, and / or myeloid leukemias, e.g., acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2;leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant [M4E] with eosinophilia), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroma; lymphomas, e.g., 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, hemangiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant, lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system Lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt 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 Sézary syndrome), and Waldenström-Mach syndrome. Lymphoplasmacytoid lymphoma (LPL) with globulinemia; myelomas, e.g., IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called inactive myeloma), solitary, plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin including fibrosarcoma and rhabdomyosarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nerves (astrocytoma) , including schwannoma); tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratocarcinoma, hematopoietic tumors of lymphoid lineage, including, but not limited to, T-cell and B-cell tumors, including T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including the small cell and cerebriform cell types;The preferred cancers include large granular lymphocyte leukemia (LGL), preferably of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of the above cancers. Preferred cancers are also listed in Fehler! Verweisquelle konnte nicht gefunden werden.;

[0054] The cancer may also be ovarian cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, thyroid cancer, liver cancer, lung cancer, stomach cancer, testicular cancer, or bladder cancer. Thus, the biomarker protein (of interest) may be an ovarian cancer biomarker protein, a breast cancer biomarker protein, a colon 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.

[0055] Exemplary cancers, cancer biomarkers with aberrant glycosylation, lectins, antibodies, and corresponding glycan modifications within the meaning of the present invention are also shown below in Fehler! Verweisquelle konnte nicht gefunden werden. Abbreviations of lectins used in the article: AAA - European eel (Anguilla anguilla) agglutinin (UniProtKB accession number: Q7SIC1), AAL - Asian mushroom lectin, ABA - mushroom (Agaricus bisporus) agglutinin, ACA - Chinese cabbage (Amaranthus caudatus) agglutinin, AHA - peanut (Arachis hypogaea) agglutinin = peanut agglutinin (PNA), AIA - jackfruit (Artocarpus integrifolia) agglutinin = jacalin, AlloA - beetle (Allomyrina dichotoma) agglutinin, AOL - Aspergillus oryzae lectin, BanLec - banana (Musa paradisiaca) lectin, BS-I - Bandeiraea simplicifolia lectin = Griffonia (Bandeiraea) simplicifolia lectin I, Con A - Concanavalin A, DBA - Dolichos biflorus agglutinin, DSA - Datura stramonium agglutinin (jacalin), ECL - Erythrina cristagalli lectin, GNA - Galanthus nivalis agglutinin, GSA I (GSL I) - Griffonia (Bandeiraea) simplicifolia lectin I, GSL II - Griffonia (Bandeiraea) simplicifolia lectin II, HHL -Hippeastrum hybrid (Amaryllis) lectin, HPA - Helix pomatia agglutinin, LBA - Phaseolus lunatus (Lima bean, LBA), LEL - Lycopersicon esculentum (Tomato) lectin, LCA - Lens culinaris agglutinin, LTA - Lotus tetragonolobus (Asparagus pea) lectin, MAA I - Cannabis sativa agglutinin I, MAA II - Cannabis sativa agglutinin II, MGBL 1 - Macrophage galactose-binding lectin 1, MGBL 2 (Macrophage galactose-binding lectin 2), NPA - Narcissus pseudonarcissus (Daffodil) lectin, PHA E - Phaseolus vulgaris agglutinin E, PHA L - Phaseolus vulgaris agglutinin L, PhoSL - Pholiota squarrosa lectin, PNA - peanut agglutinin, PSL - Pisum sativum lectin, PTA I - Psophocarpus tetragonolobus lectin I, PTA II - Psophocarpus tetragonolobus lectin II, PWM - Phytolacca americana, RCA I - Ricinus communis agglutinin I, RCA II - Ricinus communis agglutinin II, SBA - Soybean agglutinin (Glycine max agglutinin), SCA - Sambucus canadensis agglutinin = Elderberry coagglutinin (SNA), SJA - Sophora japonica agglutinin II, SNA - elderberry coagglutinin, SSA - elderberry (Sambucus sieboldiana) agglutinin, SSL - clary sage (Salvia sclarea) lectin, STL - potato (Solanum tuberosum) lectin, TJA-I -Trichosanthes japonica agglutinin I, TJA-II - Trichosanthes vulgaris agglutinin (Yamashita et al.), TVA - Triticum vulgaris agglutinin = WGA - wheat germ agglutinin, UEA - Ulex europaeus agglutinin, VVA - Vicia villosa lectin, WFA - Fuji lectin, WGA - wheat germ agglutinin = TVA - wheat germ agglutinin. The symbol "↑" which is an up arrow means an increase in the concentration of the corresponding glycan or complex (e.g., dimer, trimer, etc.). The symbol "↓" which is a down arrow means an increase in the concentration of the corresponding glycan or complex (e.g., dimer, trimer, etc.).

[0056] Table 1: Cancers, corresponding cancer biomarkers with abnormal glycosylation, lectins, and antibodies. The combinations in this table are merely examples for various cancer types. The present invention is not limited to these exemplary combinations. TIFF2025512055000002.tif154168TIFF2025512055000003.tif237168TIFF2025512055000004.tif247168TIF F2025512055000005.tif251168TIFF2025512055000006.tif243168TIFF2025512055000007.tif240168TIFF202 5512055000008.tif238168TIFF2025512055000009.tif243168TIFF2025512055000010.tif244168TIFF2025512 055000011.tif243168TIFF2025512055000012.tif254168TIFF2025512055000013.tif232168TIFF20255120550 00014.tif251168TIFF2025512055000015.tif240168TIFF2025512055000016.tif245168TIFF20255120550000 17.tif250168TIFF2025512055000018.tif241168TIFF2025512055000019.tif244168TIFF2025512055000020.t if247168TIFF2025512055000021.tif241168TIFF2025512055000022.tif247168TIFF2025512055000023.tif24 7168TIFF2025512055000024.tif249168TIFF2025512055000025.tif238168TIFF2025512055000026.tif139168

[0057] The references given in "Fehler! It doesn't matter." are as follows: TIFF2025512055000027.tif77164TIFF2025512055000028.tif243164TIFF2025512055000029.tif243164TIFF2025512055000030.tif237164 TIFF2025512055000031.tif237164TIFF2025512055000032.tif243164TIFF2025512055000033.tif236164TIFF2025512055000034.tif157164

[0058] The method of determining the glycoprofile of protein of the present invention provides information about the glycoprofile of said protein. This information is useful for diagnosing various diseases as described herein (see also Table 1). Diseases known to be characterized by proteins with altered glycoprofiles include, but are not limited to, cancer, autoimmune disease, inflammatory disease, or neurodegenerative disease.

[0059] Thus, the present invention further provides a method for diagnosing whether a subject may be at risk for or suffer from cancer, comprising: (a) contacting a sample obtained from the subject containing a cancer biomarker protein with first beads to which an antibody against the cancer biomarker protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) Signs; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the cancer biomarker protein wherein deviation of the glycoprofile from a healthy glycoprofile of the cancer biomarker protein indicates that the subject may be at risk for or may be afflicted with cancer.

[0060] Thus, the present invention further provides a method for diagnosing whether a subject may be at risk for or suffer from an autoimmune disease, comprising: (a) contacting a sample obtained from the subject containing an autoimmune disease biomarker protein with first beads to which an antibody against the autoimmune disease biomarker protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) Signs; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the autoimmune disease biomarker protein wherein deviation of the glycoprofile from a healthy glycoprofile of the autoimmune disease biomarker protein indicates that the subject may be at risk for or may be suffering from an autoimmune disease.

[0061] In the context of autoimmune disease, a glycoprofile that indicates that the subject may be at risk for or suffering from an autoimmune disease may include increased desialysation or (exposure of) GlcNAc and mannose on antibodies, which may result in activation of the "alternative lectin pathway of complement activation" by MBP (mannose binding protein).

[0062] Thus, the present invention further provides a method for diagnosing whether a subject may be at risk for or suffer from an inflammatory disease, comprising: (a) contacting a sample obtained from the subject containing an inflammatory disease biomarker protein with first beads to which an antibody against the inflammatory disease biomarker protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) Signs; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the inflammatory disease biomarker protein. wherein deviation of the glycoprofile from a healthy glycoprofile of the inflammatory disease biomarker protein indicates that the subject may be at risk for or may be suffering from an inflammatory disease.

[0063] Thus, the present invention further provides a method for diagnosing whether a subject may be at risk for or suffer from a neurodegenerative disease, comprising: (a) contacting a sample obtained from the subject containing a neurodegenerative disease biomarker protein with a first bead to which an antibody against the neurodegenerative disease biomarker protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) Signs; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the neurodegenerative disease biomarker protein wherein deviation of the glycoprofile from a healthy glycoprofile of the neurodegenerative disease biomarker protein indicates that the subject may be at risk for or may be suffering from a neurodegenerative disease.

[0064] A "healthy glycoprofile" of a biomarker protein describes the glycoprofile of that biomarker in a sample obtained from a healthy (human) subject, preferably not afflicted with cancer, an autoimmune disease, a neurodegenerative disease, and an inflammatory disease, and any other specific disease described herein related to cancer, an autoimmune disease, an inflammatory disease, or a neurodegenerative disease.

[0065] The term "lectin" as used herein refers to a carbohydrate-binding protein. Lectins are typically highly specific for one or more carbohydrate moieties (e.g., lectins react specifically with terminal glycosidic residues of other molecules, such as glycans of glycoproteins (e.g., branched sugar molecules of glycoproteins, e.g., target polypeptides within the meaning of the present invention and biomarkers as described in Table 1 herein). Lectins are generally known in the art. The skilled artisan can readily utilize which lectins can be used to bind to one or more carbohydrate moieties of interest, e.g., one or more carbohydrate moieties of glycans attached to proteins. Preferred lectins to be applied in the context of the present invention are described herein. The term "lectin" also includes Siglecs (sialic acid-binding immunoglobulin-like lectins), galectins (lectins that specifically bind β-galactoside-containing glycans), and selectins (sialyl Lewis X (SLe x ) determinant NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc, and related sialylated, fucosylated glycans. Of note, the term "lectin" as used herein also refers to glycan-binding antibodies. Thus, the term "lectin" as used herein can also encompass lectins, Siglecs, galectins, selectins, etc., and glycan-binding antibodies. Lectins can also include DNA / RNA aptamers that recognize glycans.

[0066] The lectins are core fucose, antennary fucose, Fucα1-6GlcNAc-N-Asn-containing N-linked oligosaccharides, 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, GalNAcα1-3GalNAc, GalNAcα1-3Gal, GalNAcα / β1-3 / 4Gal, α-GalNAc, GalNAcβ1-4Gal, GalNAcα1-3(Fucα1-2)Gal, Gal NAcα1-2Gal, 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), 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 biantennary, N-linked tri / tetraantennary, branched β1-6GlcNAc, Galα1-3(Fucα1-2)Galβ1-3 / 4G lcNAc, 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 (Sialyl Le a ) antigen, sialyl Lewis x (Sialyl 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 , or branched (LacNAc) n It is specific to

[0067] Carbohydrate abbreviations as 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. ME., Essentials of Glycobiology, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY), 2009).

[0068] Additionally, as used herein, the following terms are defined below. "Core fucose" means that fucose is linked via an α-glycosidic bond of its C1 atom to the C6 atom of N-acetylglucosamine; "antennary fucose" means that fucose is linked via an α-glycosidic bond of its C1 atom to the C3 atom of N-acetylglucosamine, or fucose is linked via an α-glycosidic bond of its C1 atom to the C2 atom of an adjacent fucose; "Fucα1-6GlcNAc-N-Asn-containing N-linked oligosaccharide" means an oligosaccharide having fucose linked via an α-glycosidic bond of its C1 atom to the C6 atom of N-acetylglucosamine, which is in turn linked to an asparagine via an N-glycosidic bond; "Fucα1-6 / 3GlcNAc" means that fucose is linked via an α-glycosidic bond of its C1 atom to the C6 (C3) atom of N-acetylglucosamine; "α-L-Fuc" means α-L-fucose, "Fucα1-2Galβ1-4(Fucα1-3)GlcNAc" means that a fucose is linked through an α-glycosidic bond of its C1 atom to the C2 atom of a galactose, which is linked through a β-glycosidic bond of its C1 atom to the C4 atom of an N-acetylglucosamine; and at the same time, a second fucose is linked through an α-glycosidic bond of its C1 atom to the C3 atom of the N-acetylglucosamine, "Fucα1-2Gal" means that fucose is linked via an α-glycosidic bond of its C1 atom to the C2 atom of galactose; "Fucα1-6GlcNAc" means that fucose is linked via an α-glycosidic bond of its C1 atom to the C6 atom of N-acetylglucosamine; "Manβ1-4GlcNAcβ1-4GlcNAc" means that mannose is linked through a β-glycosidic bond of its C1 atom to the C4 atom of N-acetylglucosamine, and that N-acetylglucosamine is linked through a β-glycosidic bond of its C1 atom to the C4 atom of N-acetylglucosamine, "branched N-linked hexasaccharide" means a non-linear glycan composed of several sugars linked to asparagine by N-glycosidic bonds; "Manα1-3Man" means that mannose is linked via an α-glycosidic bond of its C1 atom to the C3 atom of mannose; "α-D-Man" means α-D-mannose, "(GlcNAcβ1-4) 2-4 " means that N-acetylglucosamine is repeatedly linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond of its C1 atom, "Galβ1-4GlcNAc" means that galactose is linked via a β-glycosidic bond of its C1 atom to the C4 atom of N-acetylglucosamine; "GlcNAcα1-4Galβ1-4GlcNAc" means that N-acetylglucosamine is linked to the C4 atom of galactose via an α-glycosidic bond of its C1 atom, and that galactose is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond of its C1 atom; "N-acetylglucosamine" means the amide between glucosamine and acetic acid; "(GlcNAcβ1-4) 2-5 " means that N-acetylglucosamine is repeatedly linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond of its C1 atom, "Neu5Ac" (or sialic acid) means N-acetylneuraminic acid; "Galβ1-3GalNAc-Serine / Threonine" means that galactose is linked via a β-glycosidic bond of its C1 atom to the C3 atom of an N-acetylglucosamine, which is in turn linked to a serine / threonine; "Galα1-3GalNAc" means that galactose is linked via an α-glycosidic bond of its C1 atom to the C3 atom of N-acetylgalactosamine; "Galβ1-6Gal" means that galactose is linked via a β-glycosidic bond of its C1 atom to the C6 atom of galactose; "Galβ1-4GlcNAc" means that galactose is linked via a β-glycosidic bond of its C1 atom to the C3 atom of N-acetylglucosamine; "Galβ1-3GalNAc" means that galactose is linked via a β-glycosidic bond at its C1 atom to the C3 atom of N-acetylgalactosamine; "GalNAcα1-3GalNAc" means that an N-acetylgalactosamine is linked via an α-glycosidic bond of its C1 atom to the C3 atom of an N-acetylgalactosamine; "GalNAcα1-3Gal" means that N-acetylgalactosamine is linked via an α-glycosidic bond of its C1 atom to the C3 atom of galactose; "GalNAcα / β1-3 / 4Gal" means that N-acetylgalactosamine is linked via an α- or β-glycosidic bond of its C1 atom to the C3 or C4 atom of galactose; "α-GalNAc" means the amide between α-galactosamine and acetic acid; "GalNAcβ1-4Gal" means that N-acetylgalactosamine is linked via a β-glycosidic bond of its C1 atom to the C4 atom of galactose; "GalNAcα1-3(Fucα1-2)Gal" means that N-acetylgalactosamine is linked to the C3 atom of galactose via an α-glycosidic bond of its C1 atom, and at the same time, fucose is linked to the C2 atom of galactose via an α-glycosidic bond of its C1 atom; "GalNAcα1-2Gal" means that N-acetylgalactosamine is linked via an α-glycosidic bond of its C1 atom to the C3 atom of galactose; "GalNAcα1-3GalNAc" means that an N-acetylgalactosamine is linked via an α-glycosidic bond of its C1 atom to the C3 atom of an N-acetylgalactosamine; "GalNAcβ1-3 / 4Gal" means that N-acetylgalactosamine is linked via a β-glycosidic bond of its C1 atom to the C3 or C4 atom of galactose; "GalNAc-Ser / Thr" (or Tn antigen) means that N-acetylgalactosamine is linked to serine / threonine via an O-glycosidic bond; "Galβ1-3GalNAc-Ser / Thr" (T antigen or Thomsen-Friedenreich antigen) means that galactose is linked via a β-glycosidic bond of its C1 atom to the C3 atom of an N-acetylgalactosamine, which is in turn linked to a serine / threonine via an O-glycosidic bond; "GalNAcβ1-4GlcNAc" (or LacdiNAc) means that N-acetylgalactosamine is linked via a β-glycosidic bond of its C1 atom to the C4 atom of N-acetylglucosamine; "α2-3Neu5Ac" (or α2-3 linked sialic acid) means that N-acetylneuraminic acid is linked via an α-glycosidic bond at its C2 atom to the C3 atom of an adjacent sugar; "α2-6Neu5Ac" (or α2-6-linked sialic acid) means that N-acetylneuraminic acid is linked via an α-glycosidic bond at its C2 atom to the C6 atom of an adjacent sugar; "α2-8Neu5Ac" (or α2-8-linked sialic acid) means that N-acetylneuraminic acid is linked to the C8 atom of an adjacent N-acetylneuraminic acid via an α-glycosidic bond at its C2 atom; "Neu5Acα4 / 9-O-Ac-Neu5Ac" means that N-acetylneuraminic acid is linked via an α-glycosidic bond at its C4 atom to the C9 atom of an adjacent O-acetyl-N-acetylneuraminic acid; "Neu5Acα2-3Galβ1-4Glc / GlcNAc" means that N-acetylneuraminic acid is linked through an α-glycosidic bond of its C2 atom to the C3 atom of galactose, which is linked through a β-glycosidic bond of its C1 atom to the C4 atom of glucose or N-acetylglucosamine; "Neu5Acα2-6Gal / GalNAc" means that N-acetylneuraminic acid is linked via an α-glycosidic bond at its C2 atom to the C6 atom of galactose or N-acetylgalactosamine; "N-linked biantennary" refers to a non-linear glycan having two antennae (sugar chains) linked to asparagine by N-glycosidic bonds; "N-linked tri / tetraantennary" refers to a non-linear glycan having three / four antennae linked to asparagine by N-glycosidic bonds; "branched β1-6GlcNAc" means that an N-acetylglucosamine is linked via a β-glycosidic bond of its C1 atom to the C6 atom of an adjacent sugar; "Galα1-3(Fucα1-2)Galβ1-3 / 4GlcNAc" means that galactose is linked to the C3 atom of galactose through an α-glycosidic bond of its C1 atom, and that galactose is linked to the C3 or C4 atom of N-acetylglucosamine through a β-glycosidic bond of its C1 atom; and at the same time, fucose is linked to the C2 atom of N-acetylglucosamine through an α-glycosidic bond of its C1 atom, "Galβ1-3(Fucα1-4)GlcNAc" means that galactose is linked via a β-glycosidic bond of its C1 atom to the C3 atom of N-acetylglucosamine; and at the same time, fucose is linked via an α-glycosidic bond of its C1 atom to the C4 atom of N-acetylglucosamine; "NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc" means that N-acetylneuraminic acid is linked through an α-glycosidic bond of its C2 atom to the C3 atom of galactose, which is linked through a β-glycosidic bond of its C1 atom to the C3 atom of N-acetylglucosamine; and at the same time, fucose is linked through an α-glycosidic bond of its C1 atom to the C4 atom of N-acetylglucosamine, "Fucα1-2Galβ1-3(Fucα1-4)GlcNAc" means that a fucose is linked to the C2 atom of a galactose through an α-glycosidic bond of its C1 atom, and that galactose is linked to the C3 atom of an N-acetylglucosamine through a β-glycosidic bond of its C1 atom; and at the same time, a second fucose is linked to the C4 atom of the N-acetylglucosamine through an α-glycosidic bond of its C1 atom, "Galβ1-4(Fucα1-3)GlcNAc" means that galactose is linked via a β-glycosidic bond of its C1 atom to the C4 atom of N-acetylglucosamine; and at the same time, fucose is linked via an α-glycosidic bond of its C1 atom to the C3 atom of N-acetylglucosamine; "NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc" means that N-acetylneuraminic acid is linked through an α-glycosidic bond of its C2 atom to the C3 atom of galactose, which is linked through a β-glycosidic bond of its C1 atom to the C4 atom of N-acetylglucosamine; and at the same time, fucose is linked through an α-glycosidic bond of its C1 atom to the C3 atom of N-acetylglucosamine, "Fucα1-2Galβ1-4(Fucα1-3)GlcNAc" means that a fucose is linked to the C2 atom of a galactose through an α-glycosidic bond of its C1 atom, and that galactose is linked to the C4 atom of an N-acetylglucosamine through a β-glycosidic bond of its C1 atom; and at the same time, a second fucose is linked to the C3 atom of an N-acetylglucosamine through an α-glycosidic bond of its C1 atom, "High mannose" means a glycan containing more than three mannose units; "Cialil Lewis a " (Sialyl 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 through an α-glycosidic bond of its C2 atom, and the galactose is linked to the C3 atom of N-acetylglucosamine through a β-glycosidic bond of its C1 atom; and at the same time, fucose is linked to the C4 atom of N-acetylglucosamine through an α-glycosidic bond of its C1 atom, "Cialil Lewis x " (Sialyl 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 through an α-glycosidic bond of its C2 atom, and that galactose is linked to the C4 atom of N-acetylglucosamine through a β-glycosidic bond of its C1 atom; and at the same time, fucose is linked to the C3 atom of N-acetylglucosamine through an α-glycosidic bond of its C1 atom, "Lewis x " " x ) antigen is "Galβ1-4(Fucα1-3)GlcNAc", which means that galactose is linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond of its C1 atom; and at the same time, fucose is linked to the C3 atom of N-acetylglucosamine via an α-glycosidic bond of its C1 atom, "Sialyl-Tn antigen" is "Neu5Acα2-3 / 6GalNAc-Ser / Thr," which means that N-acetylneuraminic acid is linked via an α-glycosidic bond of its C2 atom to the C3 or C6 atom of N-acetylgalactosamine, which is in turn linked to serine / threonine via an O-glycosidic bond; "Sialyl T antigen" is "Neu5Acα2-3 / 6Galβ1-3GalNAc-Ser / Thr", which means that N-acetylneuraminic acid is linked through an α-glycosidic bond of its C2 atom to the C3 or C6 atom of galactose, which is linked through a β-glycosidic bond of its C1 atom to the C3 atom of N-acetylgalactosamine, which is linked through an O-glycosidic bond to serine / threonine; "Lewis y " " y ) antigen is "Fucα1-2Galβ1-4(Fucα1-3)GlcNAc", which means that a fucose is linked to the C2 atom of a galactose through an α-glycosidic bond of its C1 atom, which galactose is linked to the C4 atom of N-acetylglucosamine through a β-glycosidic bond of its C1 atom; and at the same time, a second fucose is linked to the C3 atom of N-acetylglucosamine through an α-glycosidic bond of its C1 atom, "Sulfated core 1 glycan" refers to a glycan based on a sulfated extended form of T antigen; "Core 2 glycan" refers to an extended glycan based on Galβ1-3(GlcNAcβ1-6)GalNAc-Ser / Thr, which has a galactose linked to the C3 atom of N-acetylgalactosamine through a β-glycosidic bond of its C1 atom, and at the same time an N-acetylglucosamine is linked to the C6 atom of N-acetylgalactosamine through a β-glycosidic bond of its C1 atom, and the N-acetylgalactosamine is linked to a serine / threonine; "Lewis a " " a ) antigen is Galβ1-3(Fucα1-4)GlcNAc, which means that galactose is linked to the C3 atom of N-acetylglucosamine via a β-glycosidic bond of its C1 atom; and at the same time, fucose is linked to the C4 atom of N-acetylglucosamine via an α-glycosidic bond of its C1 atom, "(GlcNAcβ1-4) n" means that N-acetylglucosamine is repeatedly linked to the C4 atom of N-acetylglucosamine via a β-glycosidic bond of its C1 atom, "β-D-GlcNAc" means the amide between β-D-glucosamine and acetic acid; "GalNAc" means the amide between galactosamine and acetic acid, i.e., N-acetylgalactosamine; "Gal-GlcNAc" means that galactose is linked to N-acetylglucosamine through a non-specific bond; "GlcNAc" means the amide between glucosamine and acetate, i.e., N-acetylglucosamine; "Galα1-3Gal" means that galactose is linked via an α-glycosidic bond of its C1 atom to the C3 atom of galactose; "Galβ1-3GalNAc" means that galactose is linked via a β-glycosidic bond at its C1 atom to the C3 atom of N-acetylgalactosamine; "α-Gal" means α-galactose; "α-GalNAc" means the amide between α-D-galactosamine and acetic acid; "(GlcNAc) n " means that the N-acetylglucosamine is linked to the N-acetylglucosamine through a non-specific bond; “Branch (LacNAc) n " is a branched repeating form of Galβ1,4-GlcNAc, which means a branched repeating form of galactose linked via a β-glycosidic bond at its C1 atom to the C4 atom of N-acetylglucosamine.

[0069] Lectins can be obtained from legume seeds, but also from other plant and animal sources. Lectins can contain binding sites for specific monosaccharides and oligosaccharides (e.g., glycans of glycoproteins). Lectins can agglutinate cells by binding to specific sugar residues in membrane glycoproteins. Preferably, the lectins of the present invention are selected from the group consisting of Maackia amurensis lectin II (MAA II); Concanavalin A (Con A); Aleuria aurantia lectin (AAL); Sambucus nigra (SNA-I) lectin; Wisteria floribunda lectin (WFL), as defined herein.

[0070] Further preferred lectins of the present invention are shown below in Table 1. In this context, the lectins do not necessarily have to be used in combination with the indicated antibodies or proteins, but can be seen as examples of pairs of lectins and their recognized glycan structures.

[0071] Particularly preferred lectins of the present invention are those having the following UniProtKB accession numbers (sequences according to v1 of the sequence 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, Q2F1K 8, 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.

[0072] Exemplary lectins of the present invention further include: Maackia lectin II (MAA II) is a hemagglutinin isolectin from Maackia seeds. It is a sialic acid-binding lectin that recognizes oligosaccharides containing terminal sialic acid linked to adjacent galactose residues via α2-3 linkages. It binds to the trisaccharide sequence Neu5Acα2-3-Gal-β-1-4-GlcNAc. Preferably, MAA II has SEQ ID NO: 2 (or its mature form). Concanavalin A (Con A) is a D-mannose specific lectin extracted from Canavalia ensiformis, originally a jack-bean. Preferably, Con A has SEQ ID NO: 3 or SEQ ID NO: 4 (Con A, mature form). Alar lectin (AAL) is a fucose-specific lectin extracted from Alar lectin (orange peel mushroom). Preferably, AAL has SEQ ID NO: 5 (or its mature form). Isolation of AAL has been described, for example, in (Debray et al., Kochibe et al.). Sambucus nigra (SNA-I) lectin is a Neu5Acα2-6)Gal / GalNAc specific agglutinin extracted from Sambucus nigra (European elder). Preferably, SNA-I has SEQ ID NO: 6 (or its mature form). Fuji lectin (WFL) is an agglutinin extracted from Fuji (Japanese wisteria). Preferably, WFL has SEQ ID NO: 7 (or its mature form).

[0073] Furthermore, suitable lectins within the meaning of the present invention may expressly include post-translationally processed and mature forms of lectins as disclosed herein.

[0074] As used herein, the term "beads" refers to small spherical objects, e.g. made of glass, plastic, metal, agarose, latex, metal nanoparticles or microparticles, metal oxide nanoparticles or microparticles, or magnetic materials. Thus, the first bead and / or further beads are preferably made of glass, plastic, metal, agarose, latex, metal nanoparticles or microparticles, metal oxide nanoparticles or microparticles, or magnetic materials. 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 particles or beads that contain a magnetic material or substance (e.g., iron or ferritin). Preferably, the magnetic carrier is a magnetic particle or magnetic bead (e.g., ferritin conjugate). However, for the avoidance of doubt, the magnetic carrier as referred to herein is not a solid surface, such as a plate, e.g., an ELISA plate or a microtiter plate.

[0075] As described herein, one or more additional beads include a label. The label is preferably a detectable label. Preferred labels include, but are not limited to, enzymes, radioisotopes, fluorescent proteins, fluorescent dyes, bioluminescent labels, or tags (e.g., biotin). Detectable labels can be any of a variety of types currently used in the field of in vitro diagnostics, including particulate labels that include metals such as colloidal gold, isotopes, chromophores, including fluorescent markers, biotin, luminescent markers, phosphorescent markers, and the like, as well as enzyme labels that convert a given substrate into a detectable marker, and polynucleotide tags that become evident after amplification, such as by polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, polyHRP, alkaline phosphatase, and the like, and are preferably horseradish peroxidase. For example, the label can be the enzyme alkaline phosphatase, adamantyl methoxyphosphoryloxyphenyl dioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}Decan}-4-yl)phenylphosphate disodium (CSPD), and 1,2 dioxetane substrates such as CDP and CDP-star®, or other luminescent substrates well known to those skilled in the art, for example, chelates of suitable lanthanides such as terbium (III) and europium (III), can be detected by measuring the presence or formation of chemiluminescence after conversion. The detection means are determined by the label selected. The appearance of the label or its reaction products can be achieved by the naked eye, if the label is particulate and accumulates at an appropriate level, or by instruments such as spectrophotometers, luminometers, fluorometers, etc., all according to standard practices. Thus, the label of said one or more additional beads can be detected based on optical, fluorescent, luminescent, electrochemiluminescent, and / or multianalyte profiling (xMAP) readouts or means. The label of said one or more additional beads can be detected by optical means such as absorption at a specific wavelength, or by inspection with the naked eye.The label of said one or more additional beads can be detected by fluorescence means, such as determining the emission of a fluorophore at a specific wavelength after excitation at a different, typically shorter, wavelength. The label of said one or more additional beads can be detected by electrochemiluminescence means, for example by utilizing the ELECSYS system marketed by Roche. The label of said one or more additional beads can be detected by multi-analyte profiling (xMAP), for example as described in WO 2007 / 075891. "Tags" as used herein may include, but are not limited to, affinity tags added to proteins so that they can be purified from their raw biological sources using affinity techniques, such as chitin-binding protein (CBP), maltose-binding protein (MBP), Strep tag, and glutathione-S-transferase (GST), or poly(His) tag, a widely used protein tag that binds to metal matrices; chromatography tags, such as FLAG tags, used to alter the chromatographic properties of proteins to provide different resolution across specific separation techniques; epitope tags, which are short peptide sequences selected to ensure that high affinity antibodies can be produced in many different species, such as ALFA tags, V5 tags, Myc tags, HA tags, Spot tags, T7 tags, and NE tags; fluorescent tags, such as GFP and its variants, used to give proteins a visual readout; protein tags that may allow for specific enzymatic modifications (such as biotinylation by biotin ligase) or chemical modifications (such as reaction with FlAsH-EDT2 for fluorescent imaging).

[0076] Preferably, the label is microperoxidase. As used herein, the term "microperoxidase" or "MP" refers to a heme-containing peptide portion of cytochrome c (e.g., as shown in SEQ ID NO: 11, cytochrome c from Equus caballus, NCBI Reference Sequence: 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), and preferably said 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).

[0077] A further suitable tag is biotin. Thus, the label is preferably biotin. In this embodiment, biotin can act as an anchor for the addition of a further label that can be bound to biotin. Such a further label can in principle be any label described herein, which is obviously not biotin itself. Thus, instead of directly coupling the label to the one or more further beads, the (further) label can be "indirectly" coupled to the one or more further beads by binding to biotin. Thereby, the amplification effect described herein can also be achieved. Preferably, said further label that is bound or can be bound to biotin on the one or more further beads comprises a biotin-binding moiety, such as streptavidin. It is also envisaged that the tag on the one or more further beads is a binding partner A, such as biotin, where the further label comprises a ligand B, such as streptavidin, that can specifically bind to the binding partner A. "Streptavidin" is a protein purified from the bacterium Streptomyces avidinii. Streptavidin homotetramer has an extremely high affinity for biotin (also known as vitamin B7 or vitamin H). The binding of biotin to streptavidin is approximately 10 -14 Dissociation constant (K d ), which is one of the strongest non-covalent interactions known in nature. An exemplary amino acid sequence of wild-type streptavidin is: TIFF2025512055000035.tif24164. An exemplary wild-type sequence of streptavidin is also shown in the UniProt database entry P22629, version 1, dated August 1, 1991. As used herein, for example, in the context of the methods or uses described herein, streptavidin can also include streptavidin muteins. Streptavidin muteins are disclosed, for example, in WO 2017 / 186669 or WO 2014 / 076277. The streptavidin or streptavidin muteins used in the methods and uses of the present invention can be derived from streptavidin variants that are truncated at the N-terminus or / and C-terminus. A preferred polypeptide according to the invention comprises the amino acid sequence of minimal streptavidin, starting N-terminus in the region of amino acids 10-16 and ending C-terminus in the region of amino acids 133-142. Such streptavidin mutein polypeptides preferably correspond to minimal streptavidin outside the mutated region, comprising the amino acid sequence from position Ala13 to Ser139, optionally with an N-terminal methionine residue instead of Ala13. In the present application, the numbering of the amino acid positions refers throughout to the numbering of mature wt-streptavidin (see Argarana et al., Nucleic Acids Res. 14 (1986), 1871-1882, SEQ ID NO: 1), also deposited under the accession number UniProtKB - P22629, v1 on August 1, 1991. As used herein, "streptavidin" in the context of the methods or uses described herein may also relate to other biotin-binding moieties other than streptavidin, such as proteins or aptamers that bind to biotin.

[0078] In embodiments where the label is biotin and the further label is bound to the biotin instead of being directly bound to the further bead, the actual detectable label can be added to the antibody-protein-lectin complex of step (b) after step (b), e.g. in optional step (b') or step (c).

[0079] An "antibody" as used herein is a protein comprising one or more polypeptides (including one or more binding domains, preferably antigen-binding domains) substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Preferably, an antibody to a protein whose glycoprofile is determined as described herein is not directed against a glycan attached to the protein. In other words, an antibody to a protein whose glycoprofile is determined as described herein is preferably directed against the protein itself, i.e., against an epitope within the amino acid sequence of the protein. The epitope may be a linear or conformational epitope. The epitope may be a continuous or discontinuous epitope. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. In particular, an "antibody" as used herein is typically a tetrameric glycosylated protein composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chains, called lambda and kappa, can be found in antibodies. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, with IgG being preferred in the context of the present invention. Antibodies of the present invention having an IgE constant domain or a portion thereof to which the Fcε receptor I binds are also envisaged. IgM antibodies consist of five basic heterotetrameric units with an additional polypeptide called the J chain and contain ten antigen-binding sites, whereas IgA antibodies consist of two to five basic four-chain units that can polymerize to form multivalent aggregates in combination with the J chain. For IgG, the four-chain unit is generally about 150,000 daltons.Each light chain comprises an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain comprises an N-terminal V domain (VH), three or four C domains (CH), and a hinge region. The constant domains are not directly involved in binding the antibody to the antigen, but can exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC). If the antibody is to exert ADCC, it is preferably of the IgG1 subtype, whereas the IgG4 subtype does not have the ability to exert ADCC.

[0080] The term "antibody" also includes, but is not limited to, monoclonal, monospecific, polyspecific or multispecific antibodies, such as bispecific antibodies, humanized antibodies, camelized antibodies, human antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutated antibodies, grafted antibodies, and in vitro generated antibodies, with chimeric or humanized antibodies being preferred. The term "humanized antibody" is generally defined for antibodies in which the CDRs encoding the specificity of the HC and LC have been transferred to a suitable human variable framework ("CDR grafting"). The term "antibody" also includes scFvs, single-chain antibodies, diabodies or tetrabodies, domain antibodies (dAbs), and nanobodies. In the context of the present invention, the term "antibody" is also intended to include dimeric, trimeric, or multimeric antibodies having several antigen binding sites, or bifunctional, trifunctional, or multifunctional antibodies. The term also includes antigen-binding portions. The term "antibody" can also include an FN3 scaffold, an adnectin, an affibody, an anticalin, an avimer, a bicyclic peptide, a DARPin, a Kunitz domain, an Obody, or an aptamer, e.g., a DNA, RNA, or peptide aptamer.

[0081] Preferred antibodies of the present invention include, but are not limited to, anti-PSA antibodies, anti-AFP antibodies, anti-MUC16 antibodies, anti-WFDC2 antibodies, anti-MUC1 antibodies, anti-ERBB2 antibodies, anti-CEACAM5 antibodies, anti-FUT3 antibodies, or anti-TG antibodies. Further preferred antibodies of the present invention are shown below in Fehler! Verweisquelle konnte nicht gefunden werden.

[0082] Furthermore, the term "antibody" as used herein also relates to derivatives of the antibodies (including fragments) described herein. A "derivative" of an antibody includes an amino acid sequence that has been altered by the introduction of substitutions, deletions, or additions of amino acid residues. Additionally, a derivative encompasses an antibody that has been modified by the covalent attachment of any type of molecule to the antibody or protein. Examples of such molecules include, but are not limited to, sugars, PEG, hydroxyl groups, ethoxy groups, carboxy groups, or amine groups. In effect, covalent modifications of antibodies result in, but are not limited to, glycosylation, pegylation, acetylation, phosphorylation, amidation.

[0083] As used herein, the term "specifically binds" or "to" refers to an antibody or a fragment or derivative thereof that specifically binds to a target glycoprotein or target polypeptide and does not specifically bind to another protein or polypeptide. An antibody or a fragment or derivative thereof according to the present invention binds to its respective target through the variable domain of the antibody. Typically, binding occurs when the binding affinity is greater than or equal to 10. -6 M is considered specific. Preferably, binding occurs when the binding affinity is greater than about 10 -11 ~10 -8 M(K D ), preferably about 10 -11 ~10 -9 M. If necessary, non-specific binding can be reduced by altering the binding conditions without substantially affecting the specific binding. In the case of binding of glycans to lectins, the binding affinity is preferably within the range of 10 -3~10 -6 (K D ) range. The corresponding K D Methods for measuring are known in the art and readily available to one of skill in the art.

[0084] As outlined herein, the antibody is bound to a first bead, and the lectin and label are coupled to one or more additional beads. The chemistry of coupling the antibody, lectin and label to the beads is well known to those skilled in the art. The antibody can be coupled to, for example, Protein A, Protein G or Protein L coated beads, secondary antibody coated beads or epoxy coated beads. The antibody, label and lectin can be coupled to, for example, streptavidin or fusion tag coated beads. Alternatively or additionally, the antibody can be covalently coupled to beads modified with terminal -COOH groups using amine coupling chemistry (e.g., using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or N-hydroxysuccinimide (NHS) coupling chemistry). Such beads are commercially available, for example Dynabeads® from Thermo Fisher Scientific, Waltham, Massachusetts, USA.

[0085] The present invention further relates to a kit for performing a method of diagnosing whether a subject may be at risk for or suffer from a cancer of the present invention, comprising an antibody specific for a cancer biomarker protein as defined herein and one or more lectins as defined herein.

[0086] The present invention further relates to a kit for performing a method of diagnosing whether a subject may be at risk for or suffer from an autoimmune disease of the present invention, comprising an antibody specific for an autoimmune disease biomarker protein, which is IgG, and one or more lectins as defined herein.

[0087] The present invention further relates to a kit for carrying out a method of diagnosing whether a subject may be at risk for or suffer from an inflammatory disease of the present invention, comprising an antibody specific for an inflammatory biomarker protein, which is IgG, IgA, or CRP, and one or more lectins as defined herein.

[0088] The present invention further relates to a kit for performing a method of diagnosing whether a subject may be at risk for or suffer from a neurodegenerative disease of the present invention, comprising an antibody specific for an inflammatory biomarker protein, preferably alpha-synuclein, tau protein, or amyloid beta protein and its isoforms, and one or more lectins as defined herein.

[0089] In a further aspect of the present invention, there are provided articles of manufacture and kits containing the antibody or antigen-binding portion thereof, which can be used, for example, for the therapeutic or non-therapeutic applications described above. The articles of manufacture include a container having a label. Suitable containers include, for example, bottles, vials, and test tubes. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition that includes an active agent that is effective for the therapeutic or non-therapeutic application, as described above. The active agent in the composition is an antibody or antigen-binding portion thereof. The label of the container indicates that the composition is used for a particular therapeutic or non-therapeutic application, and can also indicate instructions for either in vivo or in vitro use, such as those described above.

[0090] Kits of the present invention typically include the containers described above, as well as one or more other containers that contain materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0091] The present invention is also characterized by the following items. 1. A method for determining the glycoprofile of a protein, comprising: (a) contacting a sample containing the protein with first beads to which an antibody against the protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) Signs; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the protein A method comprising: 2. The method of claim 1, further comprising the step (d) of comparing the glycoprofile of the protein with a control glycoprofile of the protein to determine whether the glycoprofile of the protein may deviate from the control glycoprofile of the protein. 3. The method of any one of the preceding items, further comprising the step (a') of concentrating the antibody-glycoprotein complexes prior to step (b) of contacting the antibody-glycoprotein complexes with one or more additional beads. 4. The method of any one of the preceding items, further comprising the step (b') of concentrating the antibody-protein-lectin complex prior to step (c) of determining the glycoprofile of the protein. 5. The method of 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. 6. The method of item 5, wherein the cancer biomarker protein is an ovarian cancer biomarker protein, a breast cancer biomarker protein, a colon 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. 7. The method of item 6, wherein the prostate cancer biomarker protein is β-haptoglobin, TIMP-1, PSA, fPSA, or tPSA. 8. The lectin is selected from the group consisting of core fucose, antennary fucose, Fucα1-6GlcNAc-N-Asn-containing N-linked oligosaccharides, 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, GalNAcα1-3GalNAc, GalNAcα1-3Gal, GalNAcα / β1-3 / 4Gal, α-GalNAc, GalNAcβ1-4Gal, GalNAcα1-3(Fucα1-2)Gal, Gal NAcα1-2Gal, 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), 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 biantennary, N-linked tri / tetraantennary, branched β1-6GlcNAc, Galα1-3(Fucα1-2)Galβ1-3 / 4G lcNAc, 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 (Sialyl Le a ) antigen, sialyl Lewis x (Sialyl 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 , branching (LacNAc) n The method of any one of the preceding items, wherein the method is specific for 9. A method for diagnosing whether a subject may be at risk for or have cancer, comprising: (a) contacting a sample obtained from the subject containing a cancer biomarker protein with first beads to which an antibody against the cancer biomarker protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) Signs; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the cancer biomarker protein Including, a deviation of the glycoprofile from a healthy glycoprofile of the cancer biomarker protein indicates that the subject may be at risk for or may be afflicted with cancer. method. 10. A method for diagnosing whether a subject may be at risk for or suffer from an autoimmune disease, comprising: (a) contacting a sample obtained from the subject containing an autoimmune disease biomarker protein with first beads to which an antibody against the autoimmune disease biomarker protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) Signs; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the autoimmune disease biomarker protein Including, a deviation of the glycoprofile from a healthy glycoprofile of the autoimmune disease biomarker protein indicates that the subject may be at risk for or may be suffering from an autoimmune disease. method. 11. A method for diagnosing whether a subject may be at risk for or suffer from an inflammatory disease, comprising: (a) contacting a sample obtained from the subject containing an inflammatory disease biomarker protein with first beads to which an antibody against the inflammatory disease biomarker protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) Signs; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the inflammatory disease biomarker protein. Including, a deviation of the glycoprofile from a healthy glycoprofile of the inflammatory disease biomarker protein indicates that the subject may be at risk for or may be suffering from an inflammatory disease. method. 12. A method for diagnosing whether a subject may be at risk for or suffer from a neurodegenerative disease, comprising: (a) contacting a sample obtained from the subject containing a neurodegenerative disease biomarker protein with a first bead to which an antibody against the neurodegenerative disease biomarker protein is coupled, so as to form an antibody-protein complex; (b) coupling the antibody-protein complex to form an antibody-protein-lectin complex; (i) Signs; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the neurodegenerative disease biomarker protein Including, a deviation of the glycoprofile from a healthy glycoprofile of the neurodegenerative disease biomarker protein indicates that the subject may be at risk for or may be suffering from a neurodegenerative disease. method. 13. A kit for carrying out the method of item 9, comprising an antibody specific for a cancer biomarker protein as defined in item 5, and one or more lectins as defined in item 8. 14. A kit for carrying out the method of item 10, comprising an antibody specific for an autoimmune disease biomarker protein, the antibody being IgG, and one or more lectins as defined in item 8. 15. A kit for carrying out the method of item 11, comprising an antibody specific for an inflammatory biomarker protein, which is IgG, IgA, or CRP, and one or more lectins as defined in item 8. 16. A kit for carrying out the method of item 12, comprising an antibody specific for a neurodegeneration biomarker protein, preferably α-synuclein, tau protein, or amyloid β protein and its isoforms, and one or more lectins as defined in item 8. 17. The method of any one of the preceding items, wherein the first bead and the additional bead are contacted with the sample simultaneously. 18. The method of any one of the preceding items, wherein the additional bead is contacted with the sample immediately after the first bead is contacted with the sample. 19. The method of any one of the preceding items, wherein the first bead is contacted with the sample immediately after contacting the second bead with the sample. 20. The method of any one of the preceding items, wherein the first beads and the further beads are in solution during carrying out the method of any one of the preceding items. 21. The method of any one of the preceding items, wherein 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. 22. The method of any one of the preceding items, wherein the label on the additional bead is an enzyme, a radioisotope, a fluorescent protein, a fluorescent dye, a bioluminescent label, or a tag (e.g., biotin). 23. The method of any one of the preceding items, wherein the label of the additional bead is detected based on optical, fluorescent, luminescent, electrochemiluminescent, and / or multi-analyte profiling (xMAP) readout. 24. The method of any one of the preceding items, wherein a different label is used in combination for each of the one or more additional beads for each carbohydrate detected by the lectin.

[0092] It is noted that, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes one or more of such various reagents, and reference to a "method" includes reference to equivalent steps and methods known to those of skill in the art that can be modified or substituted for the methods described herein.

[0093] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0094] The term "and / or" whenever used herein includes the meaning of "and", "or", as well as "all or any other combination of the elements connected by the term".

[0095] The terms "less than" or then "greater than" do not include a specific number.

[0096] For example, less than 20 means less than the indicated number. Similarly, more than or greater than means more than or greater than a indicated number, for example, more than 80% means more or greater than 80% of a indicated number.

[0097] Throughout this specification and the claims which follow, unless the context otherwise requires, the word "comprise", as well as variations such as "comprises" and "comprising", are understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including", or, as used herein, sometimes with the term "having". As used herein, "consisting of" excludes any element, step, or ingredient not specified.

[0098] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0099] As used herein, the terms "about," "approximately," or "essentially" mean within 20%, preferably within 15%, preferably within 10%, and more preferably within 5% of a given value or range. Also, inclusive of a specific number, i.e., "about 20" includes the number 20.

[0100] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0101] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manuals, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that material incorporated by reference conflicts or is inconsistent with this specification, the present specification supersedes any such material.

[0102] The contents of all publications and patent documents cited herein are incorporated by reference in their entirety. EXAMPLES

[0103] A better understanding of the present invention and its advantages will be apparent from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0104] Example 1 In this example, the application of two types of beads to cancer diagnosis based on glycoprofiling of proteins by analysis of glycans is described (see FIG. 1). First, magnetic beads ("first beads" or beads 1) are coupled to an antibody against free PSA (fPSA) using standard bioconjugation protocols. The anti-fPSA antibody is selected to bind to an epitope on fPSA that is distant from the glycan epitope on fPSA occupied by N-glycans (Asn61). Another type of bead (non-magnetic beads; i.e., "one or more further beads" or beads 2) is coupled to a lectin capable of selectively recognizing cancer-specific glycans using standard bioconjugation protocols. The lectin is co-immobilized with the enzyme peroxidase, any other enzyme, or any tag capable of generating optical, fluorescent, chemiluminescent, and electrochemiluminescent signals. Alternatively, the enzyme or tag is attached to beads 2 already modified by lectin. In one approach, we generate beads 2 modified with different enzymes and tags, which allow for analysis in a parallel / multiplex / array format of analysis.

[0105] Beads 1 coupled to anti-fPSA antibodies are incubated with a sample containing fPSA and allowed to react for a substantial time, the complex is separated by magnetic force, and the complex attached to the magnetic beads is washed with a washing buffer. In the next step, the complex is incubated with beads 2 patterned with lectins and tags (enzymes or any other signal-generating tags), and the entire complex is separated by magnetic force and washed with a washing buffer. The assay is carried out in an ELISA plate, in solution phase in any test tube, or in a flow system of a highly automated machine. The assay can be repeated with an additional one or several lectins. In such a format, the assay shows increased sensitivity and specificity with low background signals during the analysis for the diagnosis of prostate cancer. Furthermore, a low detection limit is confirmed. The method is applicable to glycoprofiling of any other glycoproteins produced by the prostate and any other organs, and is therefore suitable for the diagnosis of other cancer types.

[0106] Example 2 The assay according to FIG. 2 uses two types of nanoparticles, namely integrated gold nanoparticles with a size of 40 nm and magnetic particles with a size of 130 nm. In this case, only fPSA antibodies were bound to the surface of 130 nm magnetic particles (MNPs). Horseradish peroxidase (HRP) and lectin were bound to the surface of gold nanoparticles with a size of 40 nm, creating a sandwich configuration in the presence of the analyte, namely fPSA protein. The sandwich approach was then used for the analysis of 110 real human serum samples. The results showed that fPSA glycoprofiling can provide much better clinical parameters compared to tPSA analysis (total PSA, a prostate cancer screening biomarker). ROC (Receiver Operating Curve) curves (Figure 2) showing the difference between tPSA (black line) and the PGI+ index (light grey line) provided the following clinical parameters: AUC 0.686 (CI95%=[0.581, 0.781], specificity=0.654, sensitivity=0.690, accuracy=0.673) for tPSA and AUC 0.803 (CI95%=[0.707, 0.890], specificity=0.712, sensitivity=0.828, accuracy=0.773) for PGI+ (glycoprofiling of fPSA).

Claims

1. 1. A method for determining the glycoprofile of a protein, comprising: (a) contacting a sample containing a protein with first beads to which an antibody against the protein is coupled so as to form an antibody-protein complex; and (b) contacting the antibody-protein complex with the lectin to form an antibody-protein-lectin complex; (i) a label that amplifies the signal generated; and (ii) Lectin with one or more additional beads, each of which is coupled to (c) determining the glycoprofile of the protein A method comprising:

2. (d) comparing the glycoprofile of the protein with the control glycoprofile of the protein to determine whether the glycoprofile of the protein may deviate from the glycoprofile of the control glycoprofile of the protein; 10. The method of claim 1, further comprising:

3. Step (a'), concentrating the antibody-glycoprotein complexes prior to step (b), contacting the antibody-glycoprotein complexes with one or more additional beads; and / or Step (b') of concentrating the antibody-protein-lectin complex prior to step (c) of determining the glycoprofile of the protein. The method of claim 1 further comprising:

4. The method of claim 1 , 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 lectin is selected from the group consisting of core fucose, antennary fucose, Fucα1-6GlcNAc-N-Asn-containing N-linked oligosaccharides, 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, GalNAcα1-3Gal, GalNAcα1-3Gal, GalNAcα / β1-3 / 4Gal, α-GalNAc, GalNAcβ1-4Gal, GalNAcα1-3(Fucα1-2)Gal, Gal NAcα1-2Gal, 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) Neu5Acα4 / 9-O-Ac-Neu5Ac, Neu5Acα2-3Galβ1-4Glc / GlcNAc, Neu5Acα2-6Gal / GalNAc, N-linked biantennary, N-linked tri / tetraantennary, branched β1-6GlcNAc, Galα1-3(Fucα1-2)Galβ1-3 / 4G lcNAc, 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 (Sialyl Le a ) antigen, sialyl Lewis x (Sialyl 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 , or branched (LacNAc) n The method of claim 1, wherein the method is specific for

6. 5. The method of claim 4, wherein the protein is a cancer biomarker protein and deviation of the glycoprofile from a healthy glycoprofile of the cancer biomarker protein indicates that the subject is at risk of or afflicted with cancer.

7. 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 colon 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.

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

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

10. 5. The method of claim 4, wherein the protein is an inflammatory disease biomarker protein and deviation of the glycoprofile from a healthy glycoprofile of the inflammatory disease biomarker protein indicates that the subject is at risk for or suffering from an inflammatory disease.

11. 5. The method of claim 4, wherein the protein is a neurodegenerative disease biomarker protein, and deviation of the glycoprofile from a healthy glycoprofile of the neurodegenerative disease biomarker protein indicates that the subject is at risk for or suffering from a neurodegenerative disease.

12. (a) an antibody specific for said cancer biomarker protein as defined in claim 7 and one or more lectins as defined in claim 5; (b) an antibody specific for said autoimmune disease biomarker protein that is IgG, and one or more lectins as defined in claim 5; (c) an antibody specific for said inflammatory biomarker protein, said antibody being IgG, IgA, or CRP, and one or more lectins as defined in claim 5; or (d) an antibody specific for said neurodegeneration biomarker protein, which is alpha-synuclein, tau protein, or amyloid beta protein and its isoforms, and one or more lectins as defined in claim 5. A kit for performing the following.

13. (a) contacting the first bead and the additional bead simultaneously with the sample; (b) contacting the first bead with the sample immediately followed by contacting the additional bead with the sample; or (c) contacting the first beads with the sample immediately after contacting the second beads with the sample; 10. The method of claim 1.

14. The first beads and the further beads are in solution during the method of claim 1; and / or 10. The method of claim 1, wherein the first bead and / or the further bead is made of glass, plastic, metal, agarose, latex, metal nanoparticles or microparticles, metal oxide nanoparticles or microparticles, or a magnetic material.

15. (a) the label on the additional bead is an enzyme, a radioisotope, a fluorescent protein, a fluorescent dye, a bioluminescent label, or a tag (e.g., biotin); and / or (b) the label of said further bead is detected based on an optical, fluorescent, luminescent, electrochemiluminescent, and / or multi-analyte profiling (xMAP) readout; and / or (c) for each of said one or more additional beads for each carbohydrate detected by the lectin, a different label is used in combination; 10. The method of claim 1.

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