WO2003040185A2

WO2003040185A2 - Anti-n-glycolyl-neuraminic acid antibodies and their use for identifying glycoproteins

Info

Publication number: WO2003040185A2
Application number: PCT/EP2002/012579
Authority: WO
Inventors: Rudolf Grimm; Carsten Brockmeyer
Original assignee: Hexal Biotech Forschungsgmbh
Priority date: 2001-11-09
Filing date: 2002-11-11
Publication date: 2003-05-15
Also published as: WO2003040185A3; EP1442063A2

Abstract

The invention relates to monoclonal antibodies which specifically bind to glycoproteins that comprise N-glycan chains with a terminal glycosidically bound N-glycolyl neuraminic acid and to hybridoma cells that produce antibodies of this type. The antibodies can be used in immunoassays in order to identify, for example, recombinant human glycoproteins.

Description

description

Anti-N-Glvkolyl-Neuraminic acid antibodies and their use for the determination of Glvkoproteins

The present invention relates to antibodies for the determination of

Glycoproteins and hybridoma cells that produce these antibodies.

The invention further relates to specific antigens that induce the formation of these antibodies. Finally, the invention relates to the use of these antibodies in immunoassays for the determination of glycoproteins.

Naturally occurring biological substances such as proteins play an important role in numerous fields of application, for example in medicine and in food technology. Such substances are either isolated from animal or vegetable sources or, not least because of the associated economic advantages, by methods of genetic engineering, in particular by overexpression of recombinant DNA in a suitable host cell.

Glycoproteins are an important group of proteins in eukaryotic cells. The glycoproteins include, for example, many serum and plasma proteins, the blood group substances, antibodies, lectins, and many enzymes, receptors and proteohormones, some of which are already used in human and veterinary medicine. Important known glycoproteins that are already used in medicine include: Erythropoietin (EPO), intrinsic factor and interferons.

Glycoproteins are proteins with a number covalently attached to the

Bound oligosaccharide polypeptide chain. These are generally shorter branched heteropolysaccharides which are linked to the peptide portion by O- or N-glycosidic bonds. The attachment of the sugar residues to the polypeptide chains with the formation of the glycoproteins is a co- and post-translational modification of the polypeptides, which is carried out in eukaryotic cells by specific glycosyltransferases and predominantly in the endoplasmic Reticulum and in the Golgi apparatus. The heteropolysaccharides bound to the polypeptide chain almost always contain

N-acetylhexosamine and hexoses (usually galactose, mannose and / or glucose) and as a terminal sugar residue N-acetylneuraminic acid. Numerous non-human eukaryotic cells and some malignant degenerate human cells are also known to produce glycoproteins in which the N-acetylneuraminic acid (Neu5Ac, NANA) to some extent, i.e. usually not more than 10% and usually not more than 5%, is substituted by another sialic acid, namely N-glycolylneuraminic acid (Neu5Gc, NGNA) (CH Hokke et al., FEBS Lett. 275: 9-14, 1990; AA Bergwerff et al "Eur. J. Biochem. 212: 639-656, 1993; Y. Fujii et al., Mol. Immunol. 19: 87-94, 1982; PL Devine et al., Cancer Res. 51 : 5826-5836, 1991). Thus, Devine et al. (loc. cit.) also a monoclonal antibody that specifically binds to mucins expressed in tumor cells with O-linked carbohydrate residues containing an N-glycolylneuraminic acid residue.

Since glycosylation is often important for the biological function of the glycoproteins, recombinantly produced human proteins, for example, should be largely identical to the corresponding naturally occurring endogenous proteins. For this reason, host cells that are able to generate glycosylation patterns that are as similar as possible to human proteins are generally used in the production by genetic engineering. The advantageous and desirable genetic engineering production of glycoproteins, which are not only functionally identical but structurally almost identical to the corresponding naturally occurring proteins, often also leads to the problem that the artificially produced product is no longer analytically or only very difficult can be distinguished from the protein in its natural form. For example, for medical research and medical examinations, it is important to be able to distinguish a drug-administered protein from a corresponding homologous endogenous protein and to be able to detect it in its presence. The problem of detecting non-endogenous substances is also increasingly occurring in cases of unauthorized substitution, for example when doping with endogenous substances Substances in which the unauthorized use of these substances must be proven.

The techniques available to date for the detection of recombinantly produced glycoproteins such as EPO in the presence of the corresponding endogenous protein are not satisfactory (cf. L. Rivier and M. Saugy, J. Toxicol., 18: 145-175, 1999, for an overview). For example, most immunoassays for the detection of EPO only measure the total amount of EPO in the serum, without being able to distinguish between exogenous and endogenous protein. The detection of exogenous protein is therefore carried out indirectly in comparison with reference groups (see e.g. M. Tanabe et al., Clin. Chem. 38: 1752-1755). However, such methods are unreliable and therefore not legally tenable. L. Wide and C. Bengtsson (Br. J. Haematol. 76: 121-127) describe an electrophoretic method which uses the different distribution of the isoforms of endogenous and recombinant EPO, the electophoretic separation being followed by an immunoassay. However, this procedure is lengthy and cannot be used for rapid tests. F. Lasne and J. de Ceaurriz (Nature, 405: 635, 2000) describe a method for the detection of recombinant EPO in urine. This method also uses charge differences between the isoforms of natural and recombinant EPO, the cause of which is considered to be a different glycosylation. The different band patterns of natural and recombinant protein are made visible by immunoblotting. However, this method is also time-consuming and can only be carried out by appropriately equipped laboratories.

The object of the present invention is therefore to rapidly and specifically detect glycoproteins expressed in non-human eukaryotic cells or in degenerate human cells, for example recombinant human glycoproteins.

This object was achieved with the monoclonal antibodies according to claim 1.

The invention therefore relates to monoclonal antibodies which bind specifically to glycoproteins, the N-glycan chains with a include terminal glycosidically bound N-glycolylneuraminic acid, and which do not bind glycoproteins with N-glycan chains that do not carry terminally glycosidically bound N-glycolylneuraminic acid, and derivatives of these antibodies.

Here and in the following, “N-glycan chains” are to be understood as the naturally occurring oligosaccharides which are linked via N-glycosidic to the polypeptide chain via asparagine residues and which have an N-acetylneuraminic acid in the form of a Neu5Ac-α- (2-> 3) at their free end. -Galactose-ß- (1- »4) - N-acetylglucosamine residues." N-Glycan chains with a terminally glycosidically bound N-glycolylneuraminic acid "are to be understood as the corresponding oligosaccharides, in which the terminal N-acetylneuraminic acid by N-glycolylneuraminic acid is substituted.

Derivatives of the antibodies according to the invention are understood to mean fragments of these antibodies and modified antibodies or antibody fragments in which the binding specificity of the antibodies or fragments is retained. Fragments of the antibodies according to the invention are, in particular, those which comprise the antigen binding region of the antibodies, for example Fab, Fab 'or F (ab') ₂ fragments. Modified antibodies or fragments are - for example chemically or enzymatically - modified antibodies or fragments, for example radioactive, for example with iodine ( ¹²⁵ l or ¹³¹ l), carbon ( ¹⁴ C) or sulfur ( ³⁵ S), or with a fluorochrome, for example fluorescein isothiocyanate ( FITC), tetramethylrhodamine isothiocyanate (TRITC) phycoerythrin (PE) or dichlorotriazine fluorescein (DTAF), labeled antibodies or antibody fragments, and conjugates of the antibodies or fragments with enzymes such as horseradish peroxidase, alkaline phosphatase, ß-D-galucose oxidase, Glucoamylase, carbonic anhydrase or acetylcholinesterase.

Glycoproteins which comprise N-glycan chains with a terminally glycosidically bound N-glycolylneuraminic acid are almost all glycoproteins with N-glycan chains which are expressed in non-human eukaryotic cells and in degenerate human cells, since the terminally occurring almost exclusively in healthy human cells N-acetylneuraminic acid of the N-glycan chain in non-human eukaryotic cells and in degenerate human cells is partially substituted by N-glycolylneuraminic acid. Therefore, in addition to terminal N-acetylneuraminic acid residues, the N-glycan chains of recombinant human glycoproteins expressed in non-human eukaryotic cells also contain terminal N-glycolylneuraminic acid residues, while the corresponding homologous endogenous glycoproteins from healthy human cells contain only N-acetylneuraminic acid residues wear. The antibodies according to the invention are now able, regardless of the protein structure, to bind to N-glycan chains with a terminally glycosidically bound N-glycolylneuraminic acid, so that, for example, recombinant human glycoproteins can be distinguished from the corresponding endogenous human proteins using the antibodies according to the invention.

Since the antibodies according to the invention can bind to the sugar structures with a terminal N-glycolylneuraminic acid regardless of the structure of the total protein, these antibodies can be used to specifically detect glycoproteins that have been expressed in non-human or degenerate human cells. For example, the antibodies according to the invention react specifically with recombinant glycoproteins produced in non-human cells, for example CHO cells, BHK cells or insect cells, for example recombinant human glycoproteins such as erythropoietin, tissue plasminogen activator and colony-stimulating factors (CSF ), Follicle-stimulating hormone, intrinsic factor, factor VII, factor VIII or interferons and interleukins, for example β-interferon (IFN-β), while they do not bind to the corresponding endogenous glycoproteins from healthy human cells.

The antibodies according to the invention bind with high specificity to oligosaccharide structures which have terminal N-glycolylneuraminic acid residues which are linked via an α- (2-3) glycosidic bond to the neighboring monosaccharide unit, a galactosyl residue. Thus, the antibodies according to the invention bind to oligosaccharides with three or more monosaccharide units which carry a Neu5Gc-α- (2-> 3) ~ galactose- ß- (1 -> 4) -N-acetylglucosamine residue. The antibodies according to the invention can belong to any class of immunoglobuins, but are preferably IgG or IgM antibodies, particularly preferably IgG antibodies, for example the IgG κ subclass.

It has surprisingly been found that the formation of antibodies which are able to bind specifically to N-glycan chains with a terminally glycosidically bound N-glycolylneuraminic acid can be induced with the aid of synthetically produced oligosaccharides which produce a terminally, glycosidically bound N- Wear glycolic neuraminic acid. The oligosaccharides themselves are incomplete antigens or haptens that are not large enough per se to elicit an immune response. Therefore, such oligosaccharides are coupled to suitable carriers, and these oligosaccharide antigens or hapten-carrier conjugates are then used for immunization.

The present invention therefore also relates to synthetic oligosaccharide antigens which comprise an oligosaccharide which is optionally coupled to a support via a spacer and which contains a terminally glycosidically bound N-glycolylneuraminic acid.

Preferred oligosaccharide antigens are those in which the terminal N-glycolylneuraminic acid is linked via an α-glycosidic bond to the adjacent monosaccharide unit, for example a D-galactosyl residue. The N-glycolylneuraminic acid is preferably linked to a D-galactosyl radical via an α- (2 → 3) -, an α- (2 → 4) - or an α- (2-> 6) -glycosidic bond.

Preferably, the oligosaccharide coupled to the support has at least three monosaccharide units, and particularly preferred oligosaccharide antigens include as the trisaccharide group a Neu5Gc-α- (2-3) -galactose-β- (1-4) -N-acetylglucosamine group of the Formula I.

Any molecule with which the oligosaccharide is capable of triggering an immune response can serve as the carrier to which the oligosaccharide is bound. Suitable carrier molecules are, for example, numerous natural or synthetic high-molecular proteins. Proteins that are usually used for the production of antigens are, for example, serum albumin, such as bovine serum albumin (BSA), human serum albumin, or rabbit serum albumin, serum globulin, thyroglobulin, hemocyanine, such as keyhole limpet hemocyanin (KLH), polylysine, polyglutaminic acid or lycine copolymers. Preferred carrier molecules are BSA and KLH. Carrier molecules which are themselves not or only weakly immunogenic are advantageously used, so that the immune response is directed preferably or exclusively against the trisaccharide structure. A preferred carrier molecule in this sense is, for example, BSA.

In order to prevent steric hindrance of the oligosaccharide carrying the antigenic determinant during immunization by the carrier and to achieve a certain freedom of movement of the oligosaccharide with respect to the carrier, the oligosaccharide structure can also be coupled to the carrier via a spacer. A “spacer” is to be understood here as bridge members which are inserted between the oligosaccharide and the carrier. The spacers are expediently bridge members with 1 to 20, in particular with 2 to 10, atoms. The spacer groups can be selected from a large number of groups known to the person skilled in the art. Suitable spacers are, for example, alkylene groups with 1 to 20 carbon atoms, preferably with 2 to 15, for example 2 to 10 carbon atoms. Alkylene groups with 3 carbon atoms have proven to be very suitable. The oligosaccharide (hapten) can be coupled to the carrier in various ways. For example, the hapten can be bound to a carrier molecule like a protein by isothiocyanate coupling, coupling with diazo compounds, coupling via amide bonds, coupling by means of reductive amination, coupling by means of glutaraldehyde or coupling by means of guanidine (see also Advances in Carbohydrate Chemistry and Biochemistry, Vol. 37, pp. 225-281, 1980; Methods in Enzymology, Vol. 1, Complex Carbohydrates, Part C, pp. 155-175, 1978; Archives of Biochemistry and Biophysics, Vol. 205, No. 2, pp. 338- 395, 1980; and GT Hermanson (ed.): Bioconjugate Techniques, S 453ff; Academic Press 1996).

The present invention also relates to trisaccharides of the general formula II

wherein Z represents a hydrogen atom or a group B-X, where X represents a reactive group via which the trisaccharide can be coupled to a support, and B represents a single bond or the above-mentioned spacer group.

The reactive group X, via which the trisaccharide can be coupled to the carrier, can be selected from a large number of reactive groups, for example -NH ₂ , -CHO, -NCS, -I, -S0 ₂ -Hal, in which Hai F, Cl , Br or I means, or -COOH.

A particularly suitable trisaccharide or hapten molecule which can be coupled to a carrier is, for example, the trisaccharide Neu5Gc-α- (2- »3 ') - lactosamine-3-aminopropylglycoside of the formula III provided with a spacer

The carriers are the above-mentioned carrier molecules known to those skilled in the art, and the haptens can be coupled as described above.

The oligosaccharides according to the invention can be prepared, for example, in accordance with the process described in the following example using the preparation of the trisaccharide of the formula III according to the invention (cf. Sherman et al. (Carbohydr. Res. 330: 445-458, 2001).

The trisaccharides thus obtained can then be coupled to a carrier, for example BSA or KLH, in a manner known per se, preferably by means of reductive amination or by means of glutaraldehyde.

The oligosaccharide antigens according to the invention (hapten-carrier conjugates) can be prepared in a manner known per se

Antibodies are used. For this purpose, mammals who have become

Production of antibodies, for example rabbits,

Guinea pigs, mice, in particular Balb / c mice and NMRI mice,

Rats, sheep, goats, cows or horses, immunized with an antigen according to the invention. Preferred antigens are those which comprise a trisaccharide of the formula I which is coupled to KLH or BSA as a carrier. to

The antigen is usually immunized with a physiological

Dilute saline to a suitable concentration and then optionally an adjuvant, for example incomplete or complete Freund's adjuvant or Gerbu adjuvant is added.

The resulting dilution or emulsion can be, for example, subcutaneously, intramuscularly, intraperitoneally, intravenously or to the mammal can also be administered in other ways. The frequency of administration can be determined in the usual way. For example, subcutaneous administration is customary every two to four weeks for 1 to 10 months, for example 1 to 3 months. After one or more "booster" immunizations, the animals are killed. The antibodies are obtained from the blood of the immunized animals by centrifuging the blood and separating the serum. If necessary, the antibodies can be removed from the serum, for example by salting out, by absorption. The formation of specific anti-N-glycolylneuraminic acid antibodies in the serum of the mammals is detected with the aid of the antigen. If the immunization was carried out, for example, with a trisaccharide of formula I coupled to KLH, then specifically the Hapten-binding antibodies were detected by testing with trisaccharides coupled to BSA, and cells from the animals with the highest antibody titer and the lowest cross-reactivity were selected for cell fusion to produce hybridoma cells.

The monoclonal antibodies according to the invention and the hybridoma cells producing them can be prepared by conventional methods known in the art (G. Koehler and C. Milstein, Nature 256: 495, 1975). For this purpose, antibody-producing cells of the selected immunized animals, for example B lymphocytes or spleen cells, are fused with myeloma cells of a suitable cell line in the presence of known fusion promoters, for example paramyxoviruses, Ca ions, lysolecithin or in particular polyethylene glycol. Preferred are myeloma cell lines which lack the gene hypoxanthine guanine phosphoribosyl transferase (HGPRT) or the enzyme thymidine kinase (TK) and which therefore contain hypoxanthine, aminophene and thymidine in a selective culture medium (HAT medium), not survive. Myeloma cell lines which do not secrete immunoglobulins or parts thereof are particularly preferred. Suitable myeloma cell lines are, for example, the PAI cell lines which are available from J.W. Stocker et al. (Res. Disclos. 217: 155; 1982).

After the fusion, the cells are divided and cultivated in selective HAT medium, only hybridoma cell lines surviving because they are from the myeloma cell lines have acquired the ability to grow in vitro and, from the antibody-producing cells of the immunized animals, the missing HGPRT or TK genes and thus the ability to survive in HAT medium.

The hybridoma cells are then unsupplemented or supplemented, for example with fetal calf serum, in conventional standard culture media, for example DMEM or RPMI 1640 medium. The cell culture supernatants are examined to determine whether they contain the desired monoclonal antibodies. As described above, the specificity of the new antibodies can be demonstrated by a positive reaction with the oligosaccharide antigen according to the invention, or with glycoproteins which comprise N-glycan chains with a terminally glycosidically bound N-glycolylneuraminic acid. If the immunization was carried out, for example, with a trisaccharide of the formula I coupled to KLH, then antibodies which bind specifically to the hapten are preferably detected by testing with trisaccharides of the formula I which are coupled to BSA. Positive clones which specifically recognize the antigen are then subsequently tested differentially for their cross-reactivity with glycoproteins which only comprise N-glycan chains with terminal N-acetylneuraminic acid, but no N-glycan chains with terminal N-glycolylneuraminic acid. Antibodies that specifically bind to glycoproteins with terminal N-glycolylneuraminic acid are selected.

Antibodies with the properties according to the invention are preferred, those of hybridoma cells obtained by fusion of mouse spleen cells with PAI cells, for example the hybridoma cells NGNA-1A3, NGNA-3H5, NGNA-6G5 obtained by fusion of spleen cells of NMRI mice with PAI cells and NGNA-7A4. The hybridoma cells NGNA-1A3, NGNA-3H5, NGNA-6G5 and NGNA-7A4 were sold on September 10, 2002 according to the Budapest contract at the "DSMZ - German Collection of Microorganisms and Cell Cultures GmbH", Mascheroder Weg 1 b, D-38124 Braunschweig, Germany, and received the accession numbers DSM ACC2573, DSM ACC2574, DSM ACC2575 and DSM ACC2576. The invention further relates to the use of the antibodies or derivatives according to the invention formed by the hybridoma cells in an immunoassay for the quantitative or qualitative determination of glycoproteins which comprise N-glycan chains with a terminally glycosidically bound N-glycolylneuraminic acid. Immunoassay is understood here to mean any determination method in which use is made of the antigen-antibody reaction between glycoproteins which comprise N-glycan chains with a terminally glycosidically bound N-glycolylneuraminic acid and the antibodies according to the invention or their derivatives. Such glycoproteins can also be determined in the presence of appropriate homologous endogenous human proteins. The determination of glycoproteins in biological material, for example human or non-human body fluids such as blood and urine, is of particular interest. Glycoproteins that can be determined and detected in this way are, for example, recombinant glycoproteins produced in non-human eukaryotic cells, for example CHO cells, BHK cells or insect cells with baculoviruses as expression systems, for example recombinant human glycoproteins such as recombinant human EPO ,

The antibodies or derivatives according to the invention can be used in any of the immunoassays known per se, for example in radioimmunoassays (RIA), enzyme immunoassays (EIA or ELISA), immunofluorescence assays (IFA) and immuno-PCR or -LCR. The assays can be used in any of the known embodiments, for example as homogeneous phase assays, as solid phase assays, as indirect or direct assays, as sandwich assays, as assays with competitive inhibition, in immunoblot methods such as "Western blotting" or as temporally Alternatively, the determination of the glycoproteins can also be carried out by measuring the shift in the elution or migration time of a complex of glycoprotein and antibody compared to its pure elution or migration time in liquid chromatographic and electrophoretic methods.

Recombinant human EPO expressed in CHO cells can be advantageously detected, for example, by adding one Examining sample first either directly or indirectly, for example via polyclonal or monoclonal anti-EPO antibodies, which are specific for EPO but cannot differentiate the target molecules, covalently or non-covalently to a suitable carrier, for example plastic surfaces made of polystyrene or polypropylene, nitrocellulose - Membranes or glass beads bind. The mixture is then incubated with monoclonal antibodies according to the invention, for example mouse antibodies. The bound antibodies are then detected with an anti-mouse antibody. To visualize the presence of recombinant EPO, an anti-mouse antibody can be used which is conjugated with alkaline phosphatase or with peroxidase or is provided with a fluorescent marker or a radioactive marker, as a result of which a measurable staining, fluorescence or chemiluminescence reaction takes place or that Radioactivity is detected.

The antibodies or the immunoassays of the present invention are therefore particularly suitable for detecting recombinant human glycoproteins in the presence of the corresponding homologous endogenous proteins. They can therefore be used to advantage in medical research and doping analysis.

Examples

Preparation of a Neu5Gc-α- (2 → 3 ') lactosamine-3-aminopropylgloscoside

The Neu5Gc-α- (2- 3 ') - lactosamine-3-aminopropylglycoside of the formula III was prepared in accordance with reaction scheme I, in which Bz is a benzoyl radical, Bn is a benzyl radical and Boc is a tert-butoxycarbonyl radical, starting from the sialyl donor Phenyl [methyl-5-acetamido-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-2-thio-D-glycero-α, β-D-galacto-2-nonulopyranoside ionate ( 2), which is available as described by A. Marra and P. Sinay (Carbohydr. Res., 187: 35-42; 1989). The starting material 2 is refluxed with Boc ₂ 0 and 4-dimethylaminopyridine (DMAP) in dry THF, as in AA Sherman et al. (Carbohydr. Res. 330: 445-458, 2001), to phenyl [methyl-4,7,8,9-tetra-0-acetyl-5- (N-tert-butoxy-carbonylacetamido) -3,5- dideoxy-2-thio-D-glycero-, ß-D-galacto-2-nonulo- pyranoside ionate (3) reacted (i), which is subsequently converted into compound 4 by complete N- and O-deacetylation in MeOH / MeONa (ii) and per-O-acetylation with Ac ₂ 0 in pyridine (iii). Compound 4 is then converted into compound 5 by elimination of the Boc group with 90% aqueous CF ₃ CO ₂ H (iv) and then with AcOCH ₂ C (0) CI in the presence of Et ₃ N at 0 ° C. in CH ₂ CI _{2 converted} to a mixture of compounds 6 and 7 (v).

3-Trifluoroacetamidopropyl- (2,3,4,6-tetra-0-benzoyl-ß-D-galactopyrano-syl) - (1- »4) -2-acetamido-3,6-di-0-benzyl-2 -deoxy-ß-D-glucopyranoside (11), prepared from a mixture of benzobromgalactose (8) and thiogalactoside (9) by reaction with 3-trifluoroacetamidopropyl-2-acetamido-3,6-di-0-benzyl-2-deoxy -ß-D-glucopyranoside (10) with the addition of MS-4 A and AgOTf in CH ₂ CI ₂ as in AA Sherman et al. (loc. cit.) is described by splitting off the benzoyl groups in MeOH / MeONa in the protected triol acceptor 3-trifluoroacetamidopropyl-2-acetamido-3,6-di-0-benzyl-2-deoxy-4-0- (6-0-benzyl -ß-D-galactopyranosyl) -ß-D-glucopyranoside (12) converted (ii). The product 12 obtained is sialylated with the compound 6 prepared as described above with NIS, TfOH, MS-3 A, MeCN 18 h at -20 ° C. regio- and stereoselectively to the trisaccharide 13, compound 14 being obtained as a by-product. Compound 13 is then hydrogenated with H ₂ over a Pd-C catalyst in MeOH (viii) and converted into the desired end product of the formula III with KOH / H ₂ 0.

The compound of formula III obtained was examined by means of ¹³ C-NMR and ¹ H-NMR. The results are shown in Table 1.

Manufacture of glyconeoconjugates

To immunize mice, the Neu5Gc-α- (2 → 3 ') -lactosamine-3-aminopropylglycoside of the formula III obtained as described above was coupled with KLH as a carrier for the production of antigen conjugates. To test the immune response and the fusion products or the monoclonal antibodies, this oligosaccharide antigen was also coupled with BSA as a carrier. The coupling to the carrier was carried out using glutaraldehyde / NaBH ₃ CN (GT Hermanson, loc. Cit.). The conjugates were purified by gel filtration (Superdex 75).

In an analogous manner, a sialyllactose-BSA conjugate was prepared using commercially available α2,3-sialyllactose.

Production of monoclonal antibodies against N-glvcosidically bound NGNA

To produce monoclonal antibodies against NGNA, a total of 9 mice (2 strains, Balb / c and NMRI) were immunized 4 x at intervals of 14 days with the KLH conjugate and adjuvant prepared as above (100 μg per mouse and injection). Complete Freund's adjuvant was used as adjuvant. The immune response was tested in the ELISA for BSA-NGNA conjugate. 14 days later, two selected NMRI mice with which the best results had been obtained received 3 injections on successive days (KLH conjugate in PBS (10 mM sodium / potassium phosphate, pH 7.2, 0.8% NaCl, 0.02 % KCI); 100 μg per mouse and injection). These follow-up injections were made with incomplete Freund's adjuvant. One day after the last injection, the mice were sacrificed and the spleens removed. The spleen cells were isolated and used to produce hybridomas with non-producing myeloma cells (PAI cells, JW Stocker et al., Res. Disclos. 21713, 1982) according to the method of Köhler and Milstein (G. Köhler and C. Milstein, op. Cit.) merged.

Positive hybridomas were again selected in the ELISA for NGNA-BSA conjugate and for recombinant EPO (ERYPO ^® , Janssen Cilag). Hybridomas which recognized the NGNA-BSA conjugate and the recombinant EPO were recloned, stabilized and cryopreserved in a manner known per se.

From 4 selected hybridoma cell lines, NGNA-1A3 (DSM ACC2573), NGNA-3H5 (DSM ACC2574), NGNA-6G5 (DSM ACC2575) and NGNA-7A4 (DSM ACC2576), monoclonal antibodies were isolated from serum-free cell culture supernatants by means of thiophilic adsorption chromatography (AT-POROS 50-OH, nanoTools Antibodytechnik GmbH and Co KG, Teningen, Germany) and subsequent gel filtration (Superdex 200). The quality of the antibodies was checked by the Lämmli method using SDS-PAGE under reducing conditions and the antibody concentration was determined by measuring the OD ₂₈₀ . The antibodies could all be assigned to the IgG1 K subclass.

ELISA

The immune response was tested and the reactivity of the antibodies against NGNA was determined by means of ELISA. For this purpose, the ELISA plates were coated with NGNA-BSA conjugate, sialyllactose-BSA conjugate or with various glycoproteins.

For coating with NGNA-BSA conjugate and sialyllactose-BSA conjugate, 0.5 μg conjugate in 50 μl 0.1 M Na ₂ HP0 per well was used (2 h, 37 ° C.). The plates were blocked with 1% BSA for 1 h and were then ready for use.

Glycoproteins with which the ELISA plates were coated were recombinant EPO (ERYPO ^®; c = 84 ug / ml), recombinant IFN-beta from CHO cells (c = 133 ug / ml), mucin (Sigma M 1778), Glycophorin MM (Sigma G7903) and Glycophorin NN (Sigma G5017). Protein dilutions (c = μg / ml in 0.1 M Na ₂ HP0 ₄ ) were prepared from the proteins mentioned. 50 μl (= 0.5 μg) were applied to each well. Dilution series of the mouse sera (in 1% BSA / PBS) were used to test the immune response in the ELISA. Cell culture supernatants of the hybridoma cells for the identification of positive fusion products in the ELISA were used undiluted. Defined antibody concentrates were used to characterize the respective antibodies in the ELISA, whereby dilution series were prepared starting from a starting dilution of 4 μg / ml.

All of the antibodies tested and the goat anti-mouse IgG HRPO (Jackson Laboratories, INC., No. 115-036-008) coupled with horseradish peroxidase (HRPO) coupled with horseradish peroxidase (HRPO) were used to prepare a ready-to-use solution with the Diluted blocking solution above.

Ready-to-use ELISA plates were incubated with serum dilutions, cell culture supernatants or antibody dilutions (50 μl per well) for 1 h at room temperature and washed 5 times with PBS. After incubation with the second antibody, goat-anti-mouse-IgG-HRPO (dilution 1: 5000, 1 h, room temperature), the mixture was washed again 5 times with PBS and with 2,2'-azino-bis (3-ethylbenzthiazolinesulfonic acid) (ABTS) developed (OD ₄₁₅ ).

Reactivity of the antibodies on the NGI-BSA and sialyllactose-BSA gene icononate

The monoclonal antibodies resulting from the hybridoma cell lines, NGNA-1A3 (DSM ACC2573), NGNA-3H5 (DSM ACC2574), NGNA-6G5 (DSM ACC2575) and NGNA-7A4 (DSM ACC2576) were analyzed by ELISA for their reactivity with NGNA -BSA and silyllactose-BSA conjugates compared. The reactivity was determined by means of dilution series (starting concentration 4 μg / ml). The results are summarized in Table 2 below. The antibody concentration required to generate a signal> twice the background is given in nanograms per milliliter. Table 2

Antibody reactivity to NGNA-BSA- and α2,3-sialyllactose-BSA-

conjugates

Clone NGNA-BSA sialyllactose-BSA

NGNA-1A3 2.0ng / ml 1000ng / ml

NGNA-3H5 2.0ng / ml 1000ng / ml

NGNA-6G5 2.0ng / ml 1000ng / ml

NGNA-7A4 2.0ng / ml 1000ng / ml

The results show that the antibodies obtained are specific for NGNA.

Reactivity of the antibodies with recombinant EPO and IFN-ß produced in CHO cells

To test the specificity, the above monoclonal antibodies were further compared by ELISA as described above with regard to their reactivity to recombinant EPO and IFN-ß protein produced in CHO cells.

The reactivity was determined via dilution series as described above. The results are summarized in Table 3 below. The antibody concentration required to generate a signal> twice the background is given in micrograms per milliliter. Table 3

Antibody reactivity to recombinant proteins produced in CHO cells

Clone EPO IFN-ß

NGNA-1A3 0.5 ug / ml 2.0 ug / ml

NGNA-3H5 0.5 ug / ml 2.0 ug / ml

NGNA-6G5 0.5 ug / ml 1,0 ug / ml

NGNA-7A4 1.0 µg / ml 2.0 µg / ml

The results show that both recombinant proteins were recognized, the recombinant EPO requiring a lower antibody concentration than the recombinant IFN-ß.

Reactivity of the antibodies with commercially available glycoproteins

The above monoclonal antibodies were further examined by ELISA as described above for their reactivity to the commercially available glycoproteins mucin type II (Sigma M 1778), glycophorin MM (Sigma G7903) and glycophorin NN (Sigma G5017). These glycoproteins contain O- and N-linked oligosaccharides with N-acetylneuraminic acid (NANA) in the α.2.3 and α2.6 positions, respectively.

The reactivity was determined as described above using dilution series (starting concentration 4 μg / ml). The results are summarized in Table 4 below. The antibody concentration required to generate a signal> twice the background is given in micrograms per milliliter. Table 4

Antibody reactivity to commercially available glycoproteins

Clone Glycophorin MM Glycophorin NN Mucin Type II

NGNA-1A3> 4 μg / ml> 4.0 μg / ml> 4 μg / ml

NGNA-3H5> 4 μg / ml> 4.0 μg / ml> 4 μg / ml

NGNA-6G5> 4 μg / ml> 4.0 μg / ml> 4 μg / ml

NGNA-7A4> 4 μg / ml> 4.0 μg / ml> 4 μg / ml

Results show that the signals are not above the background.

Claims

Expectations

1. Monoclonal antibody which binds specifically to glycoproteins which comprise N-glycan chains with a terminally glycosidically bound N-glycolylneuraminic acid, and derivatives of these antibodies.

2. The antibody of claim 1, wherein the glycoprotein is a recombinant human glycoprotein.

3. Antibody according to claim 1 or 2, which is an IgG antibody.

4. Antibody according to any one of claims 1 to 3, obtainable from a hybridoma cell as deposited under the accession number DSM ACC2573, DSM ACC2574, DSM ACC2575 or DSM ACC2576.

5. Oligosaccharide antigen which comprises an oligosaccharide which is optionally coupled to a support via a spacer and which carries a terminally glycosidically bound N-glycolylneuraminic acid.

6. Antigen according to claim 5, wherein the terminal N-glycolylneuraminic acid is linked via an α-glycosidic bond to the neighboring monosaccharide unit, in particular a galactosyl residue.

7. Antigen according to claim 5 or 6, wherein the oligosaccharide has a trisaccharide structure of the formula I.

includes.

8. The antigen of any one of claims 5 to 7, wherein the carrier is a non-immunogenic carrier.

The antigen of claim 8, wherein the non-immunogenic carrier is bovine serum albumin (BSA).

10. Trisaccharide of the general formula II

wherein Z represents a hydrogen atom or a group B-X, where X represents a reactive group via which the trisaccharide can be coupled to a support, and B represents a single bond or a spacer group.

11. trisaccharide according to claim 10 of formula III

12. Hybridoma cell producing a monoclonal antibody according to any one of claims 1 to 4, obtainable by fusion of antibody-producing cells of a mammal previously immunized with an antigen according to any one of claims 5 to 9 with a myeloma cell and subsequent selection.

13. Hybridoma cell according to claim 12, wherein the antibody-producing cell is the spleen cell of a mouse.

14. Hybridoma cell according to any one of claims 12 or 13, as deposited under the deposit numbers DSM ACC2573, DSM ACC2574, DSM ACC2575 and DSM ACC2576.

15. Use of a monoclonal antibody according to any one of claims 1 to 4 or a derivative thereof in an immunoassay for the determination of glycoproteins comprising N-glycan chains with a terminally glycosidically bound N-glycolylneuraminic acid.

16. Use according to claim 15, wherein the glycoprotein is a recombinant glycoprotein.

17. Use according to claim 15 or 16, wherein the glycoprotein is a recombinant human glycoprotein, for example erythropoietin, tissue plasminogen activator, follicle-stimulating hormone, intrinsic factor, a colony-stimulating factor (CSF), factor VII, factor VIII or is an interferon or interleukin.

18. A test kit for an immunoassay for the determination of glycoproteins comprising N-glycan chains with a terminally glycosidically bound N-glycolylneuraminic acid, containing a monoclonal antibody according to any one of claims 1 to 4 or a derivative thereof.

19. The test kit of claim 18, wherein the assay is a solid phase assay.

20. Test kit according to claim 18 or 19, wherein the assay is a sandwich assay, preferably an ELISA, RIA or IFA.

21. Use of an antigen according to any one of claims 5 to 9 for the production of antibodies.