The development of an immuno-PCR assay for quantitation of low amounts of anti-glycan human antibodies is described. The sensitivity of the assay for determination of low-affinity anti-Le IgM has been found to be 4 ng/ml (~100 pg per sample), thus being two orders of magnitude higher compared to the conventional ELISA with the same antigen.

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The level of human natural antibodies of immunoglobulin M isotype against LeC in patients with breast cancer is lower than in healthy women. The epitope specificity of these antibodies has been characterized using a printed glycan array and enzyme-linked immunosorbent assay (ELISA), the antibodies being isolated from donors’ blood using LeC-Sepharose (LeC is Galβ1-3GlcNAcβ). The isolated antibodies recognize the disaccharide but do not bind to glycans terminated with LeC, which implies the impossibility of binding to regular glycoproteins of non-malignant cells. The avidity (as dissociation constant value) of antibodies probed with a multivalent disaccharide is 10−9 M; the nanomolar level indicates that the concentration is sufficient for physiological binding to the cognate antigen. Testing of several breast cancer cell lines showed the strongest binding to ZR 75-1. Interestingly, only 7% of the cells were positive in a monolayer with a low density, increasing up to 96% at highest density. The enhanced interaction (instead of the expected inhibition) of antibodies with ZR 75-1 cells in the presence of Galβ1-3GlcNAcβ disaccharide, indicates that the target epitope of anti-LeC antibodies is a molecular pattern with a carbohydrate constituent rather than a glycan.

A repertoire of monoclonal antibodies was generated by immunization of mice with cancer-associated glycoprotein CA19.9, and two of them were selected as optimal capture and detecting counterparts for sandwich test system for detection of CA19.9. Fine epitope specificity of the antibodies was determined using printed glycan array, enzyme-linked immunosorbent assay, and inhibitory enzyme-linked immunosorbent assay. Unexpectedly, both immunoglobulins did not bind key epitope of CA19.9 glycoprotein, tetrasaccharide SiaLe A , as well as its defucosylated form sialyl Le C (known as CA-50 epitope). The antibodies were found to have different glycan-binding profiles; however, they recognized similar glycotopes with common motif Galβ1-3GlcNAcβ (Le C ), thus resembling specificity of human natural cancer-associated anti-Le C antibodies. We propose that cancer-specific glycopeptide epitope includes Galβ1-3GlcNAcβ fragment of a glycoprotein O-chain in combination with proximal hydrophobic amino acid(s) of the polypeptide chain.

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Background The α1,3‐galactosyltransferase gene‐knockout (GalT KO) mice are able to produce natural anti‐αGal antibodies apparently without any specific immunization. GalT KO mice are commonly used as a model immunological system for studying anti‐αGal responses to Gal‐positive xenografts in human. In this study, we compared the specificity of mouse and human αGal antibodies to realize the adequacy of the murine model. Methods Using hapten‐specific affinity chromatography antibodies against Galα1‐3Galβ1‐4GlcNAcβ epitope were isolated from both human and GalT KO mice blood sera. Specificity of isolated antibodies was determined using a printed glycan array (PGA) containing 400 mammalian glycans and 200 bacterial polysaccharides. Results The quantity of isolated specific anti‐Galα antibodies corresponds to a content of <0.2% of total Ig, which is an order of magnitude lower than that generally assumed for both human and murine peripheral blood immunoglobulin, with a high predominance of IgM over IgG (95% vs 5%). Analysis using a printed glycan array has demonstrated that (a) antibodies from both species bind not only the Galα1‐3Galβ1‐4GlcNAcβ epitope, but also unrelated glycans; (b) particularly, for human (but not mouse) antibodies the best binders appear to be bacterial polysaccharides; (c) the profile of mouse antibodies is broader, it is noteworthy that they recognize a variety of human blood group B epitopes and even glycans without the α‐galactosyl residue. Conclusions We believe that the mouse model should be used cautiously in xenotransplantation experiments when the fine epitope specificity of antibodies is critical.