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Epithelial Ovarian cancer has the highest mortality rate among gynaecological cancers. Altered glycosylation is associated with oncogenic transformation producing tumor-associated carbohydrate antigens. We investigated the potential of natural occurring anti-glycan antibodies in the diagnosis of ovarian cancer by using printed glycan array. Anti-glycan antibodies bound to 203 chemically synthesized printed glycans were detected via biotin-streptavidin fluorescence system in serum of women with normal operative findings (healthy controls; n=24) and non-mucinous borderline or ovarian cancer of various FIGO stages (n=33). Data were validated measuring blood group associated di-, tri and tetra- saccharide antigens on known ABO blood groups. Anti-glycan antibodies demonstrated high reproducibility (rc>0.9). Cluster analysis identified repetitive patterns of specific core carbohydrate structures: 11 N-linked glycans, 3 O-linked glycans, 2 glycosphingolipids. Biomarker detection revealed 24 glycans including P1 (Galα1-4Galβ1-4GlcNAcβ; p<0.001) significantly discriminating between (low-) malignant tumors and healthy controls. Comparable sensitivity and specificity with tumor marker CA125 was achieved by a panel of multivariate selected and linear combined anti-glycan antibody signals (79.2% and 84.8%, respectively). Our findings demonstrate the potential of glycan arrays in the development of a new generation of biomarkers for ovarian cancer.

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Natural antibodies, part of the innate immunity system, are produced at strictly regulated levels in normal sera without immunization and thus are part of the innate immune system. The best studied natural antibodies are those directed against blood group antigens A and B and xeno-antigens including glycolylneuraminic acid containing Hanganutziu-Deicher (HD) glycolipid. Abnormal levels of anti-glycan antibodies were found in a number of pathologies. In many cases pathological antibodies are known to bind gangliosides. The genesis of anti-glycan antibodies in healthy humans and the reasons for their changes in pathologies are poorly understood. With a growing interest in their diagnostic applications, it is important to determine the carbohydrate structures that are recognized by antibodies present in the circulation of healthy individuals. We tested a large number of healthy donors using a printed glycan array (PGA) in a microchip format. The PGA contained ~300 glycans, representing mostly normal mammalian structures of glycoproteins and glycolipids, and many of the structures presented are biologically relevant sialylated motifs. As revealed by PGA, the sera interacted with at least 70 normal human glycans. With only few exceptions, antibodies recognizing sialosides have not been identified. Moderate levels of antibodies and moderate variability were observed in the case of SiaT n and its glycolyl variant. Unexpectedly, we found minimal antibody titer directed against Neu5Gcα and the trisaccharide Neu5Gcα2-6Galβ1-4GlcNAc, although this form of neuraminic acid does not occur naturally in humans. Antibodies recognizing sialosides in unnatural β-configuration have been detected and confirmed Springer's paradigm that circulating antibodies represent a reaction against bacteria. Gram-negative bacteria contain LPS with βKDN and/or βKDO which are very close analogs of Neu5Ac that are found in β-connected form. Antibodies against the biantennary N-glycan chain, (Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα)2-3,6-Manβ1-4GlcNAcβ1-4GlcNAc were never observed and similarly we never saw antibodies directed against the SiaLe(a)/SiaLe (x) motifs. Anti-sialoglycan antibodies can be masked with gangliosides: for example, we observe about a five times higher level of anti-GD3 in purified total IgG compared to the same concentration of total Ig in the composition of native serum. For several antibodies we observed anomalous binding in diluted sera, namely, the signals towards sialylated glycans were increased in the PGA if diluted sera were used.