A general and accessible approach to enrichment and characterisation of natural anti-Neu5Gc antibodies from human samples

<i>N</i>-Glycolylneuraminic acid (Neu5Gc) is a non-human sialic acid which is presented on the surface of human cells following uptake from dietary sources. Antibodies against Neu5Gc have implications for many aspects of human health such as inflammation, xenograft rejection and cancer. However, current methods to detect and study anti-Neu5Gc antibodies require complex synthesis of glycan structures, animal handling expertise, or access to expensive reagents and equipment. Here, we outline a simple workflow to enrich and detect anti-Neu5Gc antibodies from small volume human serological samples. This strategy involves a micro-scale affinity purification step, followed by an indirect ELISA detection step which uses CMAH-transfected human cells as a source of Neu5Gc-containing human glycans in their native context. Parental wild type cells are also used as a paired Neu5Gc-negative control. Using this workflow, Neu5Gc-specific antibodies could be enriched from intravenous immunoglobulin (IVIG) and individual plasma specimens from ten healthy donors. Anti-Neu5Gc antibodies were detected in all donors, regardless of age or sex. The lysate ELISA assay was also sufficiently sensitive to observe reproducible individual differences in the anti-Neu5Gc reactivity of each donor specimen. Importantly, despite this individual variation, enriched antibodies from all donor specimens bound effectively to Neu5Gc-containing glycans presented on the surface of whole human cells, highlighting the potential physiological relevance of these antibodies.