Standards for protein glycoprofiling

Abstract

The present invention relates to a method for relativizing signals by using a neoglycoprotein as a standard for glycoproteins, which comprises a streptavidin molecule bound through biotin to at least one predefined glycan determinant comprising a glycan structure (A), and to the corresponding use of the neoglycoprotein, which allows a statement of whether a particular glycan structure (A) is present on a protein of interest. Applications in the diagnosis of diseases such as cancer, autoimmune diseases, or inflammatory diseases are disclosed.

Description

[Technical field]

[0001] TECHNICAL FIELD OF THEINVENTION The present invention relates to a method for relativizing signals by using neoglycoproteins as standards for glycoproteins, the neoglycoproteins comprising streptavidin molecules bound via biotin to at least one, preferably four predefined glycan determinants comprising glycan structure (A), and the corresponding uses of the neoglycoproteins, which allow a statement of whether a particular glycan structure (A) is present on a protein of interest. Applications in the diagnosis of diseases such as cancer, autoimmune diseases, or inflammatory diseases are disclosed. [Background technology]

[0002] 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.

[0003] 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 particular glycan structures in assays such as enzyme-linked immunosorbent assays (ELISA), enzyme-linked lectin assays (ELLA), and magnetic ELLA (MELLA).

[0004] However, the signals obtained by applying these methods need to be relative to (positive) controls or standards to allow a reasonable statement of whether a particular glycan structure is present or not, or to quantify it in a sample. Commercially available glycoprotein standards contain only one glycan structure that is not identical to the glycan structure of interest, i.e., they cannot be used to evaluate whether a disease-related glycan structure is present on a protein of interest. Thus, protein standards containing a specific glycan structure are not readily available. One possibility to obtain such a control is to conjugate the glycan structure of interest to the protein of interest. However, this is complex, laborious, and expensive. Moreover, it must be repeated for every new combination of the glycan structure of interest and the protein of interest.

[0005] Thus, there is a continuing need for reliable, inexpensive and versatile glycoprotein standards, which the present invention aims to address. Summary of the Invention

[0006] This need is addressed by the subject matter as defined in the claims and embodiments described herein.

[0007] The inventors have surprisingly found that, in order to relativize the signal obtained from the determination of the glycan structure (A) suspected to be present on the protein of interest (1), it is not necessary to provide such a modified (glyco)protein of interest, but instead a neoglycoprotein can be used as a standard. This standard provides a signal (2) obtained from the determination of said glycan structure (A) actually contained by the neoglycoprotein. The neoglycoprotein comprises a streptavidin molecule bound through biotin to at least one predefined glycan determinant comprising said glycan structure (A). Thus, streptavidin can be seen as a scaffold for the neoglycoprotein, which itself serves as a standard for relativization. As shown in the examples, streptavidin loaded with a biotinylated glycan structure (A) can be used as a standard.

[0008] The present invention therefore relates to a method for relativizing a signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest, comprising a step of comparing the signal obtained from determining the glycan structure (A) suspected to be present on the protein of interest with a signal (2) obtained from determining the glycan structure (A) actually contained by a neoglycoprotein, the neoglycoprotein comprising a streptavidin molecule bound via biotin to at least one predefined glycan determinant comprising the glycan structure (A), thereby relativizing the signal (1) to the signal (2) or vice versa.

[0009] The present invention also relates to a method for relativizing a signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest, comprising a step of comparing the signal obtained from determining the glycan structure (A) suspected to be present on the protein of interest with a signal (2) obtained from determining the glycan structure (A) actually contained by a neoglycoprotein serving as a standard, the neoglycoprotein comprising a streptavidin molecule bound through a biotin-streptavidin interaction to at least one predefined glycan determinant comprising the glycan structure (A), wherein relativizing comprises a step of comparing signal (1) with signal (2) from the standard, thereby allowing signal (2) to relate to the information obtained by signal (1), thereby relativizing signal (1) to signal (2) or vice versa.

[0010] The present invention further comprises: of a neoglycoprotein comprising a streptavidin molecule bound via biotin to at least one predefined glycan determinant that actually contains a glycan structure (A) suspected to be present on a protein of interest; To relativize the signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest to the signal (2) obtained from determining the glycan structure (A) actually contained by said neoglycoprotein. Use, the neoglycoprotein comprises a streptavidin molecule bound to at least one predefined glycan determinant comprising the glycan structure (A) through a streptavidin binding molecule; Regarding use.

[0011] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: of a neoglycoprotein to serve as a standard, comprising a streptavidin molecule bound via biotin to at least one predefined glycan determinant that actually contains the glycan structure (A) suspected to be present on the protein of interest; To relativize the signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest to the signal (2) obtained from determining the glycan structure (A) actually contained by said neoglycoprotein. Use, the neoglycoprotein comprises a streptavidin molecule bound through a biotin-streptavidin interaction to at least one predefined glycan determinant comprising the glycan structure (A); and relativizing comprises comparing signal (1) with a signal (2) from a standard, thereby allowing signal (2) to correlate with the information obtained by signal (1); Regarding use.

[0012] Relativizing may involve comparing signal (1) with signal (2) from a standard, thereby making it possible to correlate signal (2) with the information obtained by signal (1). Advantageously, in such a comparison, signal (1) is relativized with respect to said signal (2), or vice versa.

[0013] Relativizing may include comparing the signal obtained from determining the glycan structure (A) suspected to be present on the protein of interest with the signal (2) obtained from determining the glycan structure (A) actually contained by the neoglycoprotein, where (i) if signal (1) is lower than signal (2), it indicates that the suspected glycan structure (A) is not present on the protein of interest, or (ii) if signal (1) is equal to or higher than signal (2), it indicates that the suspected glycan structure (A) is present on the protein of interest.

[0014] The relativizing may further comprise comparing a signal (1) obtained from determining the glycan structure (A) suspected to be present on the protein of interest with a signal (2) obtained from determining a concentration series of the glycan structure (A) actually contained by the neoglycoprotein.

[0015] The concentration series may include concentrations corresponding to a predetermined threshold concentration above which the glycan structure (A) is known to be present on the protein of interest.

[0016] The signal may be a signal intensity.

[0017] The signal (1) and the signal (2) can be obtained by enzyme-linked immunosorbent assay (ELISA), enzyme-linked lectin assay (ELLA), magnetic ELLA (MELLA), preferably ELLA or MELLA.

[0018] Glycan structure (A) consists of core fucose, antennary fucose, N-linked oligosaccharides containing Fucα1-6GlcNAc-N-Asn, Fucα1-6 / 3GlcNAc, α-L-Fuc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Gal, Fucα1-6GlcNAc, Manβ1-4GlcNAcβ1-4GlcNAc, branched N-linked hexasaccharides, Manα1-3Man, α-D-Man, (GlcNAcβ1-4) 2-4 , Galβ1-4GlcNAc, GlcNAcα1-4Galβ1-4GlcNAc, (GlcNAcβ1-4) 2-5, Neu5Ac (sialic acid), Galβ1-3GalNAc-serine / threonine, Galα1-3GalNAc, Galβ1-6Gal, Galβ1-4GlcNAc, Galβ1-3GalNAc, 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 may be selected from the group consisting of:

[0019] The protein of interest can be a cancer biomarker protein, an autoimmune disease biomarker protein, or an inflammatory disease biomarker protein.The cancer biomarker protein can 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 gastric cancer biomarker protein, a testicular cancer biomarker protein, or a bladder cancer biomarker protein.The prostate cancer biomarker protein can be β-haptoglobin, TIMP-1, PSA, fPSA, or tPSA.

[0020] The presence of said glycan structure (A) may be indicative of cancer. [Brief description of the drawings]

[0021] 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:

[0022] [Figure 1]An exemplary scheme of an exemplary embodiment of the present invention is illustrated. MAA-II lectin and optionally a blocking agent such as BSA are physically adsorbed to the bottom of an ELISA plate well. Magnetic particles with co-immobilized anti-streptavidin antibodies and horseradish peroxidase (HRP) are used to selectively probe the analyte (in this case, a glycan-streptavidin bioconjugate) and then applied to the lectin-biorecognition interface. A light signal is generated using o-phenylenediamine and hydrogen peroxide to form the colored product 2,3-diaminophenazine and detected by a common ELISA reader (e.g., λ=450-490 nm). [Diagram 2] Figure 2A illustrates the gradient values ​​during the pH study for streptavidin (left column) and MAA-II lectin (right column), and Figure 2B shows sensorgrams illustrating the immobilization of three different ligands in pH 4.0 acetate buffer using a CM5 chip. [Diagram 3] Illustrates SCK (single cycle kinetics) analysis of anti-streptavidin Ab on a streptavidin-modified CM5 chip. [Figure 4] (A) A bare Au SPR chip with a prism on one site, (B) modified with a self-assembled monolayer of 11-mercaptoundecanoic acid or (C) carboxymethyl-dextran, creating 2D and 3D matrices, respectively, are illustrated. Because the amplitude of the evanescent wave decays exponentially (red arrow), 3D matrices, which together with a high concentration of negative charges, steric hindrance, and a higher probability of rebinding during the dissociation phase, were less suitable for observing the sandwich configuration, i.e., MAA-II / neoglycoprotein / Ab preparation. [Diagram 5] Illustrated is the assay workflow used in the 2D configuration, including schematics of the surface after each step (Figure 5A), as well as sensorgrams of neoglycoprotein (glycoconjugate) capture and anti-streptavidin antibody binding analysis (Figure 5B). [Figure 6]Illustrates the model situation on a planar surface (e.g., SPR chip, A) when the linker density is the same near the Au surface and on the interface (in a 2D configuration, i.e., no diffusion barrier in the matrix). However, the situation is different for a spherical interface (e.g., MNP, B). NTA analysis of three samples (C), showing successful immobilization and interaction with neoglycoproteins. [Figure 7] NTA analysis of unmodified MNPs (thick line) and MNPs+Ab (with excess antibody, light line) is shown, where a clear increase in the peak maximum caused by the antibody immobilized on the surface was observed. [Figure 8] Illustrated is the MS analysis of neoglycoprotein (protein standard) showing the highest intensity for the approximately 13 kDa peak (single streptavidin monomer). [Figure 9-1] Illustrated is an MS analysis showing other peaks (with lower intensity compared to FIG. 8). (A); a peak at approximately 1030 Da corresponding to biotinylated glycan is not detectable in the sample with pure streptavidin (B), but is present in all other samples, even at lower glycan / streptavidin ratios and after the desalting procedure, meaning that glycans are present on streptavidin (C), a fact already confirmed using SPR. The intensity of this peak increases with increasing glycan / streptavidin ratio (D). Using ELLBA, the optimal ratio for preparing neoglycoproteins with saturating density of glycans was found in a competitive configuration with unconjugated and biotinylated MAA-II lectin (E), with a ratio of 1+5 or higher being found to be optimal (F). [Figure 9-2] This is a continuation of Figure 9-1. [Figure 9-3] This is a continuation of Figure 9-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description of the Invention The present invention is described in detail below and further illustrated by the accompanying examples and figures.

[0024] When available, the standard used in the prior art is typically a protein of interest that is labeled with a glycan structure (A) suspected to be present on the protein of interest (of interest). Thus, the standard in the prior art is the protein of interest itself, which contains the glycan structure (A). However, synthesizing such a standard is complicated, time-consuming, and expensive. Therefore, the present invention aims to provide a reliable, inexpensive, and versatile glycan structure or glycoprotein standard for relativizing the signal obtained from determining the glycan structure suspected to be present on the protein of interest.

[0025] The inventors have surprisingly found that in order to relativize the signal obtained from the determination of the signal (1) obtained from the determination of the glycan structure (A) suspected to be present on the protein of interest, it is not necessary to provide such a modified (glyco)protein of interest, but instead the neoglycoprotein can be used as a standard. This signal (2) obtained from the determination of the glycan structure (A) on the neoglycoprotein allows the relativization of the signal (1). The neoglycoprotein comprises a streptavidin molecule bound through biotin to at least one predefined glycan determinant comprising said glycan structure (A). Thus, streptavidin can be seen as a scaffold for the neoglycoprotein, to which the glycan structure (A) of interest can be coupled.

[0026] As shown in the examples, streptavidin loaded with biotinylated glycan structure (A) can be used as a standard. This is the first study to develop a glycoconjugate or exemplary neoglycoprotein as described herein, consisting of streptavidin molecules bound to up to four biotinyl-glycans to form a neoglycoprotein with four glycans on a protein / streptavidin scaffold. At the same time, the concept of the present invention allows the preparation of any kind of neoglycoprotein with a defined glycan structure (A) present on the surface of streptavidin using biotinylated glycans. Such neoglycoproteins with (up to) four glycans may then be used as protein standards to determine said glycan structure (A), for example in the MELLA technology disclosed in WO 2019 / 185515 A1. In an exemplary embodiment, such neoglycoproteins can bind to a lectin and an anti-streptavidin antibody simultaneously, i.e., in a sandwich configuration, to provide a light signal, which is believed to be a signal against which signals from analysis of a sample can be relativized using Glycanostics' MELLA protocol for prostate cancer diagnosis (among other things) (Figure 1).

[0027] The present invention therefore relates to a method for relativizing a signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest, comprising a step of comparing the signal obtained from determining the glycan structure (A) suspected to be present on the protein of interest with a signal (2) obtained from determining the glycan structure (A) actually contained by a neoglycoprotein, the neoglycoprotein comprising a streptavidin molecule bound via biotin to at least one predefined glycan determinant comprising the glycan structure (A), thereby relativizing the signal (1) to the signal (2) or vice versa.

[0028] The present invention also relates to a method for relativizing a signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest, comprising a step of comparing the signal obtained from determining the glycan structure (A) suspected to be present on the protein of interest with a signal (2) obtained from determining the glycan structure (A) actually contained by a neoglycoprotein serving as a standard, the neoglycoprotein comprising a streptavidin molecule bound through a biotin-streptavidin interaction to at least one predefined glycan determinant comprising the glycan structure (A), wherein relativizing comprises comparing signal (1) with signal (2) from the standard, thereby allowing signal (2) to relate to the information obtained by signal (1), thereby relativizing signal (1) to signal (2) or vice versa.

[0029] The present invention further comprises: of a neoglycoprotein comprising a streptavidin molecule bound via biotin to at least one predefined glycan determinant that actually contains a glycan structure (A) suspected to be present on a protein of interest; To relativize the signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest to the signal (2) obtained from determining the glycan structure (A) actually contained by said neoglycoprotein. Use, the neoglycoprotein comprises a streptavidin molecule bound to at least one predefined glycan determinant comprising the glycan structure (A) through a streptavidin binding molecule; Regarding use.

[0030] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: of a neoglycoprotein to serve as a standard, comprising a streptavidin molecule bound via biotin to at least one predefined glycan determinant that actually contains the glycan structure (A) suspected to be present on the protein of interest; To relativize the signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest to the signal (2) obtained from determining the glycan structure (A) actually contained by said neoglycoprotein. Use, the neoglycoprotein comprises a streptavidin molecule bound through a biotin-streptavidin interaction to at least one predefined glycan determinant comprising the glycan structure (A); and relativizing comprises comparing signal (1) with a signal (2) from a standard, thereby allowing signal (2) to correlate with the information obtained by signal (1); Regarding use.

[0031] "Relativizing" can be seen as describing the process of comparing one signal (1) with another signal (2), thereby providing information about how signal (1) relates to signal (2). Signal (2) can be seen as a standard in the context of the present invention. In other words, signal (2) allows the information obtained by signal (1), such as signal intensity, to be contextualized or related. Thus, signal (2) can act as a positive control, thereby providing information about the signal required to consider signal (1) positive. Alternatively or additionally, signal (2) can be not only a positive control, but also the result of a concentration series, thereby allowing quantification of the amount of glycan structure (A) by comparing (relativising) signal (1) with a series of signals (2) at different concentrations. In this context, the method and use of the present invention can also be described as glycoprofiling of a protein of interest.

[0032] In line with the above, "relativising" may include comparing signal (1) with signal (2) from a standard, which allows signal (2) to relate the information obtained by signal (1). Advantageously, in such a comparison, signal (1) is relativised to said signal (2), or vice versa.

[0033] 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" 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 a protein of interest.

[0034] The information provided by signal (1) and signal (2) can provide information on whether glycan structure (A) is present on the protein of interest. For this purpose, signal (1) (obtained from determining the glycan structure (A) suspected to be present on the protein of interest) is compared (relativised) with standard / signal (2) (obtained from determining the glycan structure (A) actually contained by the neoglycoprotein). If signal (1) is equal to or higher than signal (2), it indicates that the glycan structure (A) is considered to be present on the protein of interest. Thus, relativising can include comparing signal (1) obtained from determining the glycan structure (A) suspected to be present on the protein of interest with signal (2) obtained from determining the glycan structure (A) actually contained by the neoglycoprotein, and if signal (1) is lower than signal (2), it indicates that the suspected glycan structure (A) is not present on the protein of interest.

[0035] Otherwise, if signal (1) is lower than signal (2), it indicates that the glycan structure (A) is considered not to be present on the protein of interest. Thus, relativizing can include comparing signal (1) obtained from determining the glycan structure (A) suspected to be present on the protein of interest with signal (2) obtained from determining the glycan structure (A) actually contained by the neoglycoprotein, and if signal (1) is equal to or higher than signal (2), it indicates that the suspected glycan structure (A) is present on the protein of interest.

[0036] However, the method and use of the present invention is not limited to a qualitative statement. In contrast, when signal (1) is compared with a series of signals (2) determined with a series of different concentrations of neoglycoprotein, a quantitative analysis of the amount of glycan structure (A) suspected to be present on the protein of interest in a sample is possible. Thus, relativeization (in the method and use of the present invention) can include comparing signal (1) obtained from determining the glycan structure (A) suspected to be present on the protein of interest with signal (2) obtained from determining the concentration series of the glycan structure (A) actually contained by the neoglycoprotein.

[0037] A "concentration series" as described herein refers to a data set of signals (2) obtained from determining glycan structure (A) at two or more different concentrations of neoglycoprotein. The data set can be used to provide or calculate a standard curve, for example by linear regression, that allows a conclusion from the signal (intensity) (1) to the actual level or amount of glycan structure (A) in the sample. The concentration series can also allow for setting a predetermined threshold value that provides a signal (intensity) at which the glycan structure (A) is considered to be present on the protein of interest. In a different way, the concentration series can be used to set a baseline (threshold) of data points at which no neoglycoproteins have been added to the sample to be analyzed (in the concentration series). Thus, the concentration series can also include concentrations that correspond to a predetermined threshold concentration above which the glycan structure (A) is known to be present on the protein of interest.

[0038] "Neoglycoprotein" as used herein refers to a streptavidin molecule to which at least one and up to four, i.e. one, two, three or four, preferably four, biotinylated glycan structures (A) are non-covalently bound by biotin-streptavidin interactions. Thus, the neoglycoprotein is bound through biotin-streptavidin interactions. Streptavidin thereby serves as a scaffold to which the desired glycan structure (A) can be coupled. This provides a versatile and rapidly available neoglycoprotein standard. Thus, the glycan structure (A) is preferably added to streptavidin in excess, for example at a molar ratio of at least 4:1 (glycan structure (A):streptavidin), at least 5:1, at least 7.5:1, or at least 10:1. Preferably, the glycan structure (A) is added to the streptavidin in a molar ratio between 4.5:1 and 5.5:1, most preferably in a molar ratio of 5:1.

[0039] "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: TIFF2024530669000001.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.

[0040] Streptavidin may have an amino acid sequence that is substantially identical to that of SEQ ID NO: 1. Streptavidin as used herein may have an amino acid sequence that has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. Streptavidin as used herein may consist of an amino acid sequence that has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

[0041] Generally, as used herein, the term "percent identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to the degree to which two or more sequences or subsequences are the same. Two sequences are "identical" if they have the same sequence of amino acids or nucleotides over the region being compared. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity over a specified region, or if not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window or a specified region, as measured using one of the following sequence comparison algorithms, or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 30 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500, or 1000 or more nucleotides (or 20, 50, 200, or more amino acids) in length.

[0042] The percentage of sequence homology or sequence identity can be determined herein, for example, using the program BLASTP, version blastp 2.2.5 (November 16, 2002) (see Altschul et al., Nucleic Acids Res, 1997). In this embodiment, the percentage of homology is preferably determined by alignment of the entire polypeptide sequence, including the propeptide sequence, using the wild-type protein scaffold as a reference in a pairwise comparison (matrix: BLOSUM 62; gap cost: 11.1; 10 -3 It is calculated as a percentage of the number of resulting "positives" (homologous amino acids) in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0043] "Glycan" or "glycan structure (A)" refers to a compound consisting of glycosidically linked monosaccharides, and can also refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, glycoRNA (see, e.g., Flynn et al, Cell, 184(12):3109-3124), or proteoglycan, even if the carbohydrate is only a monosaccharide or oligosaccharide.

[0044] The glycan structure (A) bound to streptavidin in the neoglycoprotein described herein is advantageously biotinylated. "Biotinylation" is the process of covalently attaching biotin to a protein, nucleic acid, or other molecule, in particular to the glycan structure (A) as described herein. Various methods for biotinylating the glycan structure (A) are known to those skilled in the art. For example, biotinylation can be based on reductive amination, in which a primary amine is coupled to an aldehyde to form an imine, or a hydrazone if the primary amine is present as a hydrazide group. This imine (hydrazone) is then reduced to a secondary amine, which stabilizes the linkage formed (see scheme below). Using this reaction procedure, biotin-LC-hydrazide can be easily coupled to the reducing end of any carbohydrate to form a stable biotin-labeled product. See, for example, Grun et al. (2006), Analytical Biochemistry, 354(1):54-63, which is incorporated by reference in its entirety. TIFF2024530669000002.tif93161

[0045] Biotin may also be coupled to the glycan by a polyethylene glycol linker, such as a triethylene glycol (PEG3) spacer between the glycan and biotin. An exemplary glycan structure (A) is 3'-sialyllactosamine-PEG3-biotin (single arm, approximately 1100 Da), which is a biotinylated 3'-sialylated N-acetyllactosamine (LacNAc=LN=Galβ1,4GlcNAc) with a β-linked triethylene glycol (PEG3) spacer between the glycan and biotin. Such biotinylated glycans are commercially available, for example, from Sussex Chemicals, Ottawa, Canada.

[0046] The present invention is not limited to any particular glycan structure (A). In contrast, the modular construction of neoglycoproteins allows for non-covalent attachment of virtually any glycan structure (A) to streptavidin, the scaffold of the neoglycoproteins described herein. Exemplary glycan structures (A) include 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 These include, but are not limited to:

[0047] 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).

[0048] 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 linked via an O-glycosidic bond to a serine / threonine; "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 N-acetylglusosamine is linked to the C6 atom of an adjacent sugar via a β-glycosidic bond at its C1 atom; "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 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; "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.

[0049] "Signal" as used herein refers to information obtained by determining the (suspected) glycan structure (A) on a protein of interest. This information is typically a signal intensity, for example, absorption at a particular wavelength or fluorescence emission at a particular wavelength. The signal intensity preferably depends on the amount of glycan structure (A) present on the protein of interest. Thus, the signal can also be seen as providing information about the glycoprofile of the protein of interest.

[0050] The neoglycoproteins described herein serve as standards in the methods and uses of the present invention. They relate to determining the glycan structure (A) that provides signal (1) or signal (2). "Determining glycan structure (A)" as used herein relates to any method suitable for assaying whether a particular glycan structure (A) is present on a protein of interest, preferably suitable for determining the amount of glycan structure (A) (in a sample). In other words, determining glycan structure (A) can also be seen as a method of glycoprofiling a protein of interest suspected to have glycan structure (A) thereon. Thus, this method provides a signal (signal (1)) to be relativized or a signal (signal (2)) that is the basis for relativization. Suitable methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), enzyme-linked lectin assay (ELLA), magnetic ELLA (MELLA), preferably ELLA or MELLA. Thus, signal (1) and signal (2) can be obtained by enzyme-linked immunosorbent assay (ELISA), enzyme-linked lectin assay (ELLA), magnetic ELLA (MELLA), preferably ELLA or MELLA. Preferably, signal (1) and signal (2) are obtained under the same conditions, including the same reagents at the same concentrations. However, the present invention is not limited to these methods and can include any other assay method involving glycan analysis.

[0051] ELISA can be seen as a starting point for various similar or ELISA-like assays. ELISA is based on the specific interaction between an immunoglobulin and a molecule of interest, such as a glycan structure (A) suspected to be on a protein of interest, which allows the generation of a signal only in the presence of the molecule of interest, and the signal (intensity) corresponds to the concentration of the molecule of interest in the sample being analyzed. Assays such as ELISA, ELLA, and MELLA are known to those skilled in the art. Various ELISA variations, such as sandwich ELISA, competitive ELISA, and reverse ELISA, are further known in the art. In the context of the present invention, the primary immunoglobulin, i.e. the immunoglobulin that binds to the molecule of interest, binds to the glycan structure (A) suspected to be on the protein of interest. Thus, the primary immunoglobulin, preferably an antibody or a fragment thereof, specifically binds to the glycan structure (A) of interest. In ELLA, the primary immunoglobulin is replaced by a lectin that specifically binds to the glycan structure (A). Magnetic ELLA (MELLA) is the result of a further development of ELLA, described in WO 2019 / 185515 A1, the entirety of which is incorporated herein by reference. The readout of these assays, e.g., signal intensity, can be seen as signal (1) or signal (2) in the context of the present invention. Typically, the readout is the result of an enzymatic reaction of an enzyme coupled to a primary immunoglobulin, a protein of interest (the protein backbone of a glycoprotein of interest), or a second immunoglobulin that binds to a lectin. Alternatively, the enzyme can be (directly) coupled to a lectin.Frequently used readouts include, but are not limited to, OPD (o-phenylenediamine dihydrochloride), which turns amber, for detecting HRP (horseradish peroxidase), often used as a conjugated protein; TMB (3,3',5,5'-tetramethylbenzidine), which turns blue when detecting HRP and turns yellow after the addition of sulfuric or phosphoric acid; ABTS (2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), which turns green when detecting HRP; or PNPP (p-nitrophenyl phosphate disodium salt), which turns yellow when detecting alkaline phosphatase. In addition to these colorimetric readouts, radioactive labels, fluorescent labels, electrochemical labels, chemiluminescent labels, colorimetric approaches, or even label-free formats (e.g., SPR) can also be used. Another example is 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red reagent) for detecting hydrogen peroxide (H2O2) or peroxidase activity. In the presence of peroxidase, Amplex® Red reagent reacts with H2O2 in a 1:1 stoichiometry to produce the red fluorescent oxidation product, resorufin, which has excitation and emission maxima at approximately 571 nm and 585 nm.

[0052] 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.

[0053] 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.

[0054] Further preferred lectins of the present invention are shown in Table 1 below.

[0055] Particularly preferred lectins of the present invention are those having the following UniProtKB accession numbers: P0DKL3, P02866, P18891, O04366, A0A218PFP3, Q945S3, Q00022, Q6YNX3, Q71QF2, P02872, P18670, Q2UNX8, Q8L5H4, A0A089ZWN7, P05045, P19588, P83410, P17931, P56470, P24146, Q41263, Q39990, Q2F1K8, G9M5T0, B3 XYC5, 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.

[0056] 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).

[0057] Furthermore, suitable lectins within the meaning of the present invention may expressly include post-translationally processed and mature forms of lectins as disclosed herein.

[0058] 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.

[0059] Preferred antibodies of the present invention include, but are not limited to, anti-PSA antibody, anti-AFP antibody, anti-MUC16 antibody, anti-WFDC2 antibody, anti-MUC1 antibody, anti-ERBB2 antibody, anti-CEACAM5 antibody, anti-FUT3 antibody, or anti-TG antibody, etc. Further preferred antibodies of the present invention are shown in Table 1 below.

[0060] 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.

[0061] The protein of interest is not limited to a particular protein. In contrast, and due to the versatility of neoglycoproteins, the method and use of the present invention can be applied to assay whether a particular glycan structure (A) is present on a protein of interest. However, the protein of interest is preferably a glycoprotein, or, stated differently, a protein suspected of having a glycan structure (A). Since the presence or absence of a glycan structure (A) on a protein of interest may be important for the diagnosis or prognosis of a disease, the protein of interest is preferably a protein whose glycoprofile is associated with a disease. The term "glycoprotein" (or "glycosylated protein") as used herein means a protein that contains one or more N-, O-, S-, or C-covalently linked carbohydrates of various types, ranging, for example, from monosaccharides to branched polysaccharides (including their modifications, 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 C-C bond.

[0062] 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.

[0063] As already described herein, the presence of a particular glycan structure (A) on a protein of interest may be associated with the diagnosis of a particular disease, such as cancer, an autoimmune disease, or an inflammatory 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 proteins of interest and glycans that are indicative of disease are exemplified in Table 1, as well as antibodies or lectins that bind to particular glycan structures. Thus, the methods and uses of the present invention can be used in the diagnosis of diseases, such as cancer, an autoimmune disease, or an inflammatory disease. Thus, the presence of said glycan structure (A) may be indicative of disease, such as cancer, an autoimmune disease, or an inflammatory disease.

[0064] In this context, the protein of interest is preferably a cancer biomarker protein, an autoimmune disease biomarker protein, or an inflammatory disease biomarker protein.

[0065] 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.

[0066] 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.

[0067] 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;Preferably, the cancers include large granular lymphocyte leukemia (LGL) 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 shown in Table 1.

[0068] 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 (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.

[0069] Exemplary cancers, cancer biomarkers with aberrant glycosylation, lectins, antibodies, and corresponding glycan modifications within the meaning of the present invention are also shown in Table 1 below. Table 1: Abbreviations of lectins used in cancer, corresponding cancer biomarkers, lectins, and antibodies with aberrant glycosylation: AAA - European eel (Anguilla anguilla) agglutinin (UniProtKB accession number: Q7SIC1), AAL - Amaranthus caudatus agglutinin, ABA - mushroom (Agaricus bisporus) agglutinin, ACA - Amaranthus caudatus agglutinin, AHA - 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 (Lens) agglutinin, LTA - Lotus tetragonolobus (Asparagus pea) lectin, MAA I - Macrophage agglutinin I, MAA II - Macrophage 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 (Phaseolus) agglutinin E, PHA L - Phaseolus vulgaris agglutinin L, PhoSL - Pholiota squarrosa (Sugitake mushroom) lectin, PNA - Peanut agglutinin, PSL - pea (Pisum sativum) lectin, PTA I - winged bean (Psophocarpus tetragonolobus) lectin I, PTA II - winged bean II, PWM - pokeweed (Phytolacca americana), RCA I - castor bean (Ricinus communis) agglutinin I, RCA II - castor bean agglutinin II, SBA - soybean agglutinin (soybean (Glycine max) agglutinin), SCA - elderberry (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 japonica agglutinin (Yamashita et al.), TVA - wheat (Triticumvulgaris 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.).

[0070] 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. TIFF2024530669000003.tif124169TIFF2024530669000004.tif243169TIFF2024530669000005.tif244169TIF F2024530669000006.tif253169TIFF2024530669000007.tif239169TIFF2024530669000008.tif251169TIFF202 4530669000009.tif252169TIFF2024530669000010.tif237169TIFF2024530669000011.tif251169TIFF2024530 669000012.tif240169TIFF2024530669000013.tif250169TIFF2024530669000014.tif238169TIFF20245306690 00015.tif247169TIFF2024530669000016.tif246169TIFF2024530669000017.tif247169TIFF20245306690000 18.tif246169TIFF2024530669000019.tif248169TIFF2024530669000020.tif250169TIFF2024530669000021.t if243169TIFF2024530669000022.tif248169TIFF2024530669000023.tif243169TIFF2024530669000024.tif24 8169TIFF2024530669000025.tif244169TIFF2024530669000026.tif240169TIFF2024530669000027.tif161169

[0071] The references shown in Table 1 are as follows: TIFF2024530669000028.tif77164TIFF2024530669000029.tif244164TIFF2024530669000030.tif244165TIFF2024530669000031.tif238164 TIFF2024530669000032.tif238165TIFF2024530669000033.tif244164TIFF2024530669000034.tif237164TIFF2024530669000035.tif157164

[0072] The present invention further relates to the following items: 1. A method for relativizing a signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest, comprising: comparing the signal obtained from determining the glycan structure (A) suspected to be present on the protein of interest with the signal (2) obtained from determining the glycan structure (A) actually contained by a neoglycoprotein, which preferably serves as a standard; Including, the neoglycoprotein comprises a streptavidin molecule bound via biotin, or for the avoidance of doubt via a biotin-streptavidin interaction, to at least one predefined glycan determinant comprising the glycan structure (A); Preferably, the relative determination involves comparing signal (1) with a signal (2) from a standard, thereby allowing signal (2) to correlate with the information provided by signal (1); Thereby, signal (1) is relativized to signal (2), or vice versa. method. 2. Relativizing Comparing the signal (1) obtained from determining the glycan structure (A) suspected to be present on the protein of interest with the signal (2) obtained from determining the glycan structure (A) actually contained by the neoglycoprotein. Including, (i) if signal (1) is lower than signal (2), indicating that the suspected glycan structure (A) is not present on the protein of interest; or (ii) if signal (1) is equal to or higher than signal (2), it indicates that the suspected glycan structure (A) is present on the protein of interest; Method of item 1. 3. Relativizing Comparing the signal (1) obtained from determining the glycan structure (A) suspected to be present on the protein of interest with the signal (2) obtained from determining the concentration series of the glycan structure (A) actually contained by the neoglycoprotein. The method of item 1, 4. The concentration series is a predetermined threshold concentration above which the glycan structure (A) is known to be present on the protein of interest; The method of item 3, including a concentration corresponding to 5. A streptavidin molecule bound via biotin to at least one predefined glycan determinant that actually contains the glycan structure (A) suspected to be present on the protein of interest, preferably of a neoglycoprotein that serves as a standard; To relativize the signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest to the signal (2) obtained from determining the glycan structure (A) actually contained by said neoglycoprotein. Use, the neoglycoprotein comprises a streptavidin molecule bound to at least one predefined glycan determinant comprising the glycan structure (A) via a streptavidin binding molecule, preferably via a biotin-streptavidin interaction; Preferably, the relative determination involves comparing signal (1) with a signal (2) from a standard, thereby allowing signal (2) to correlate with the information provided by signal (1). use. 6. Relativizing Comparing the signal (1) obtained from determining the glycan structure (A) suspected to be present on the protein of interest with the signal (2) obtained from determining the glycan structure (A) actually contained by the neoglycoprotein. Including, (i) if signal (1) is lower than signal (2), indicating that the suspected glycan structure (A) is not present on the protein of interest; or (ii) if signal (1) is equal to or higher than signal (2), it indicates that the suspected glycan structure (A) is present on the protein of interest; Use of item 5. 7. Relativizing Comparing the signal (1) obtained from determining the glycan structure (A) suspected to be present on the protein of interest with the signal (2) obtained from determining the concentration series of the glycan structure (A) actually contained by the neoglycoprotein. Use of item 6, including: 8. The concentration series is a predetermined threshold concentration above which the glycan structure (A) is known to be present on the protein of interest; Use of item 7, including the corresponding concentrations. 9. The method or use of any one of items 1 to 8, wherein the signal is a signal intensity. 10. The method or use of any one of the preceding items, wherein the signal (1) and the signal (2) are obtained by enzyme-linked immunosorbent assay (ELISA), enzyme-linked lectin assay (ELLA), magnetic ELLA (MELLA), preferably ELLA or MELLA. 11. The glycan structure (A) 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 or use of any one of the preceding items, selected from the group consisting of: 12. The method or use of any one of the preceding items, wherein the protein of interest is a cancer biomarker protein, an autoimmune disease biomarker protein, or an inflammatory disease biomarker protein. 13. The method or use of item 13, 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. 14. The method or use of item 14, wherein the prostate cancer biomarker protein is β-haptoglobin, TIMP-1, PSA, fPSA, or tPSA. 15. The method or use of any one of the preceding items, wherein the presence of the glycan structure (A) is indicative of cancer. 16. The method or use of any one of the preceding items, wherein the protein of interest is obtained from a sample obtained from a subject. 17. The method or use of item 16, wherein the sample is a saliva sample, a serum sample, a tissue sample, a blood sample, a urine sample, a lymph sample, a nasopharyngeal wash sample, a sputum sample, a mouth swab sample, a throat swab sample, a nasal swab sample, a bronchoalveolar lavage sample, or a bronchial secretion sample. 18. The method or use of item 16 or 17, wherein the subject is a mammal, preferably a human.

[0073] 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.

[0074] 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.

[0075] 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 that term".

[0076] The terms "less than" or then "greater than" do not include a specific number.

[0077] 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.

[0078] 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.

[0079] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] The contents of all publications and patent documents cited herein are incorporated by reference in their entirety. EXAMPLES

[0084] 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.

[0085] Example 1 material and method chemicals Unconjugated streptavidin (~55 kDa, non-glycosylated), MAA-II (~130 kDa), and anti-streptavidin antibody (~150 kDa) were purchased from Vector Labs, US. Glycan 3'-sialyllactosamine-PEG3-biotin (single arm, ~1100 Da) was obtained from Sussex Research, Canada. Desalting columns (MWCO=7000 Da) were purchased from Thermo, US. All common chemicals such as buffer components were purchased from Sigma Merck, US. Ultrapure deionized water (G=0.055 μS) was used for all experiments. All buffers were filtered using a 0.22 μm sterile filter before use.

[0086] Surface Plasmon Resonance (SPR) All reagents used for SPR were purchased from GE Healthcare, including HBS-P+ (buffer 10x; BR-1006-71), coupling kit (BR100050), EDC (0.4 M), NHS (0.1 M), ethanolamine hydrochloride (1 M; pH 8.5). NaOH (50 mM; BR-1003-58) was used for regeneration, and acetate buffer pH 4.0 (BR-1003-51) was used for coupling. SPR assays were performed on a BiacoreX100 (GE Healthcare) using sensor chips CM5 (29-1496-04) or Au chips modified with 11-mercaptoundecanoic acid (UV / VIS 5 mM in ethanol, Sigma Merck, US; incubated overnight at RT) at 25 °C under a constant flow rate of 30 μL / min. The instrument was run using the original SW Biacore X100 Control Software.

[0087] Nanoparticle Tracking Analysis (NTA) All samples were diluted in 0.1 M PB (pH 7.4, 0.22 μm filtered) and measured in continuous flow (a total volume of 500 μl was prepared for a single run). Measurement concentrations were found by pre-testing the ideal particles per frame value (20-100 particles / frame) as suggested. The following settings were made according to the manufacturer's software manual for the NanoSight NS300: the detection threshold was determined to include as many particles as possible, with the limit that 10-100 red crosses were counted. The counting of blue crosses was limited to a minimum. The autofocus was adjusted to avoid obscuring particles. For each measurement, five 60-second videos were taken under the following conditions: cell temperature: 25°C; syringe speed: 10 μl / min. After capture, the videos were analyzed by the built-in NanoSight Software NTA 3.1 Build 3.1.46 with a detection threshold = 10.

[0088] Results and Discussion Preparation of streptavidin-glycan neoglycoproteins Lyophilized streptavidin powder was resuspended in sterile 0.1 M PB (pH 7.4) to a concentration of 1 mg.ml -1 The concentration of the biotinylated glycan was obtained. For a known concentration of biotinylated glycan, these two solutions were mixed in a 1+5 molar ratio for 1 h at 37°C with gentle mixing (500 rpm). Finally, the neoglycoprotein was purified of redundant glycans using a 7k MWCO Zeba Spin desalting column (previously equilibrated with PB) at 3000 rpm for 1 min. The neoglycoprotein was purified to a concentration of c = approx. 0.9 mg.ml -1 and stored at 4°C for the remainder of the experimental work.

[0089] SPR binding analysis. In the first run, a carboxymethyl-dextran CM5 SPR chip was used to find the optimal immobilization pH value, after which the ligand was immobilized on the sensor surface. The optimal pH in all cases was 4.0, based on the gradient values ​​during preconcentration of the ligand on the sensor interface for different pH values ​​(Figure 2A). The pI values ​​of streptavidin and MAA-II lectin are 5.0 and 4.7, respectively, while similar values ​​are predicted for the neoglycoprotein as well. In all three cases, i.e., for streptavidin, neoglycoprotein, and MAA-II lectin, amine coupling (EDC / NHS protocol) was used for activation and ethanolamine blocking (Figure 2B). To complete the sandwich configuration, anti-streptavidin antibody (Ab) and MAA-II lectin should bind simultaneously to the neoglycoprotein. However, likely due to steric hindrance, the rather high density of negative charges, and the layer thickness / distance from the prism / gold interface (as well as a combination of these factors), we were only able to observe Ab binding to the streptavidin-modified CM5 chip during single cycle kinetics (SCK) (Figure 3). For the remaining experiments, the higher the concentration of sample in SCK, the more negative the response usually became, even during lectin binding with neoglycoprotein-modified chips (with MAA-II, SNA-I, and WGA, all of which should bind to sialylated / negatively charged glycan moieties).

[0090] To confirm the successful preparation of the sandwich (MAA-II / glycan-streptavidin / antibody), a 2D chip was prepared by immersing a bare Au chip in a 5 mM ethanolic solution of 11-mercaptoundecanoic acid (RT, in the dark, overnight). The advantage of this approach is that essentially all negative charges (carboxy groups) are localized on the surface and removed after immobilization / blocking. The entire sandwich preparation and assay workflow is shown in Figure 5A.

[0091] A detailed overview of the conditions applied at each step is summarized in Table 2 below.

[0092] Table 2: Overview of the individual steps applied in the sandwich preparation with SPR binding assay: type of molecule, conditions and results, as shown in Figure 4. All RUs are calculated after subtraction of the blank (flow cell 1, FC1) from the detection cell (FC2). Regeneration of the surface is optional and does not apply to the Glycanostics MELLBA assay, however, the surface could not be fully regenerated using only 50 mM NaOH. TIFF2024530669000036.tif81170

[0093] NTA analysis of the modification and enrichment process of MNPs For the NTA analysis observing the modification of magnetic nanoparticles (MNPs, 130 nm COOH-terminated, dextran-coated nanomag), all three samples, i.e. bare MNPs, Ab-modified MNPs (MNPs+Ab) and neoglycoprotein-enriched Ab-modified MNPs (MNPs+Ab+C), were treated equally, whether with a specific chemical / component or with PB in the blank case, and therefore all three samples were separated in the same way / same number of times. Using a spherical interface for biorecognition yielded a higher signal in this case, because the ligand density decreases with longer linker molecules, reducing the steric hindrance of MELLBA compared to the planar surface used for the SPR experiments above (Figure 6A and B; hypothesis, published elsewhere). The results from NTA are shown in Figure 6C. After modification / enrichment, the modified MNPs were more likely to form a sediment at the bottom of the test tube as a result of their increased diameter. For NTA analysis, each sample was diluted 500-fold in sterile PB.

[0094] Unmodified (but comparable times separated) MNPs almost exclusively gave rise to one single peak with d≦140 nm, whereas after Ab conjugation (amine coupling, 10 min, RT) there were two major fractions: (i) ≦120 nm (slightly smaller compared to unmodified particles) and about 20% of (ii) ≦150 nm. After incubation of these particles with purified neoglycoprotein, at least four different fractions could be observed, with even larger aggregates of about 220 nm. The hydrodynamic diameter (d H The decrease in d is caused by a "compression" of the dextran matrix around the immobilized ab if the binding capacity of the MNPs is not saturated during the immobilization process (hypothesis). Otherwise, d H increases as in Figure 7. The important aspect of this experiment is that d H 20 nm increase in β-glycan, a value influenced by the fact that the glycans (trisaccharides and PEG3 linkers) are heavily hydrated and quite mobile.

[0095] MALDI-TOF MS and ELLBA for detecting glycan content Fully glycosylated protein standards were prepared with various streptavidin + biotinyl-glycan ratios (i.e., ratios of the number of molecules per volume of 1+0, 1+1, 1+2, 1+3, 1+4, 1+5, 1+6, and 1+7). A MALDI-TOF mass spectrometer (Bruker, USA) and a previously optimized protocol (with 2,5-dihydroxyacetophenone (DHA) matrix) were used to detect mass fragments (m / z parameters) for the ionized sample components as previously described elsewhere. Figure 9 shows the mass spectra in detail, i.e., the streptavidin fragments in all samples (A) differ by about 13 kDa, which is the mass of one subunit in a homotetramer of about 55 kDa. The most intense peak is always at about 13 kDa as well (Figure 8), while the other peaks have much lower intensity. Only samples pre-incubated with biotinyl-glycans (all except 1+0, i.e., bare streptavidin, Figure 9B) also showed the presence of a peak at m / z = approx. 1300 Da, which is a 3'-sialylated glycan derivative (Figure 9C). Moreover, the intensity of this peak increased with increasing glycan / streptavidin ratio (Figure 9D). However, since streptavidin can only bind a maximum of four biotins, it is important to use a reasonable glycan / streptavidin ratio to maintain full saturation of the protein standards with these glycans.

[0096] For this purpose, an enzyme-linked lectin binding assay (ELLBA) was used to find the full saturation ratio. The assay configuration is illustrated in Figure 9E left, where a protein standard is immobilized on the bottom of an ELISA plate well, incubated with unconjugated MAA-II (specific for 2,3-sialic acid / Gal, which can effectively block all available glycan epitopes), and then incubated with biotinylated MAA-II, which carries several biotin molecules and binds to the free biotin binding sites on the unsaturated streptavidin standard. Streptavidin-peroxidase is then used to generate an optical signal (OPD / hydrogen peroxide). Streptavidin-HRP binds only to the free biotin molecules present on the biotinylated MAA-II, thus correlating with the amount of glycan molecules present on the streptavidin (Figure 9E right).

[0097] conclusion Neoglycoproteins (protein standards) generated by attachment of biotinylated glycans to streptavidin were simultaneously recognized by MAA-II lectin and by anti-streptavidin antibodies. This unexpected finding indicates that such neoglycoproteins can be applied as protein standards for ELISA-like format analyses, including MELLA.

[0098] Example 2 Experimental work has shown that glycoprotein standards, also referred to herein as neoglycoproteins (standards) or standards of the present invention, such as the standard applied in Example 1 above, are stable for at least one week when stored at 4° C. Such standards can be prepared in a highly reproducible manner, i.e., RSD of 8.17% for preparation of glycoprotein standards in 17 independent preparation batches within 18 weeks.

Claims

1. 1. A method for relativizing a signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest, comprising: a) determining the glycan structure (A) suspected to be present on a protein of interest and providing a signal (1); and b) comparing the signal (1) obtained from determining the glycan structure (A) suspected to be present on the protein of interest in step a) with the signal (2) obtained from determining the glycan structure (A) actually contained by a neoglycoprotein, which serves as a standard. Including, the neoglycoprotein comprises a streptavidin molecule bound to at least one predefined glycan determinant comprising the glycan structure (A) through a biotin-streptavidin interaction; Relativizing involves comparing signal (1) with signal (2) from a standard, thereby allowing signal (2) to relate to the information provided by signal (1); Thereby, signal (1) is relativized to signal (2), or vice versa. method.

2. Relativizing includes: (i) if signal (1) is lower than signal (2), it indicates that the suspected glycan structure (A) is not present on the protein of interest; or (ii) if signal (1) is equal to or higher than signal (2), it indicates that the suspected glycan structure (A) is present on the protein of interest; 10. The method of claim 1.

3. To relativize Comparing the signal (1) obtained from determining the glycan structure (A) suspected to be present on the protein of interest with the signal (2) obtained from determining the concentration series of the glycan structure (A) actually contained by the neoglycoprotein.

2. The method of claim 1, comprising:

4. The concentration series is a predetermined threshold concentration above which the glycan structure (A) is known to be present on the protein of interest; 4. The method of claim 3, comprising a concentration corresponding to:

5. of a neoglycoprotein to serve as a standard, comprising a streptavidin molecule bound via biotin to at least one predefined glycan determinant that actually contains the glycan structure (A) suspected to be present on the protein of interest; To relativize the signal (1) obtained from determining a glycan structure (A) suspected to be present on a protein of interest to the signal (2) obtained from determining the glycan structure (A) actually contained by the neoglycoprotein. Use, the neoglycoprotein comprises a streptavidin molecule bound to at least one predefined glycan determinant comprising the glycan structure (A) through a biotin-streptavidin interaction; Relativizing involves comparing signal (1) with signal (2) from a standard, thereby allowing signal (2) to correlate with the information provided by signal (1); use.

6. Relativizing includes: (i) if signal (1) is lower than signal (2), it indicates that the suspected glycan structure (A) is not present on the protein of interest; or (ii) if signal (1) is equal to or higher than signal (2), it indicates that the suspected glycan structure (A) is present on the protein of interest; The use according to claim 5.

7. To relativize Comparing the signal (1) obtained from determining the glycan structure (A) suspected to be present on the protein of interest with the signal (2) obtained from determining the concentration series of the glycan structure (A) actually contained by the neoglycoprotein.

7. The use according to claim 6, comprising:

8. The concentration series is a predetermined threshold concentration above which the glycan structure (A) is known to be present on the protein of interest; 8. The use according to claim 7, comprising a concentration corresponding to:

9. 6. The method of claim 1 or the use of claim 5, wherein the signal is signal intensity.

10. 6. The method of claim 1 or the use of claim 5, wherein the signals (1) and (2) are obtained by enzyme-linked immunosorbent assay (ELISA), enzyme-linked lectin assay (ELLA), or magnetic ELLA (MELLA).

11. The glycan structure (A) is a branched N-linked hexasaccharide containing core fucose, antennary fucose, Fucα1-6GlcNAc-N-Asn, Fucα1-6 / 3GlcNAc, α-L-Fuc, Fucα1-2Galβ1-4(Fucα1-3)GlcNAc, Fucα1-2Gal, Fucα1-6GlcNAc, Manβ1-4GlcNAcβ1-4GlcNAc, 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 , branching (LacNAc) n 6. The method of claim 1 or the use of claim 5, selected from the group consisting of:

12. 6. The method of claim 1 or the use of claim 5, wherein the protein of interest is a cancer biomarker protein, an autoimmune disease biomarker protein, or an inflammatory disease biomarker protein.

13. 13. The method or use of claim 12, wherein the cancer biomarker protein is an ovarian cancer biomarker protein, a breast cancer biomarker protein, a colorectal cancer biomarker protein, a pancreatic cancer biomarker protein, a prostate cancer biomarker protein, a thyroid cancer biomarker protein, a liver cancer biomarker protein, a lung cancer biomarker protein, a stomach cancer biomarker protein, a testicular cancer biomarker protein, or a bladder cancer biomarker protein.

14. 14. The method or use of claim 13, wherein the prostate cancer biomarker protein is beta-haptoglobin, TIMP-1, PSA, fPSA, or tPSA.

15. The method of claim 1 or the use of claim 5, wherein the presence of the glycan structure (A) is indicative of cancer.

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