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

N-glycolylneuraminic acid (Neu5Gc) is a non-human sialic acid which is presented on the surface of human cells following uptake from dietary sources. This is widely hypothesised to drive the production of anti-Neu5Gc antibodies, which have implications for many aspects of human health. However, current methods to detect and study anti-Neu5Gc antibodies rely on complex synthesis of glycan structures, animal handling expertise, or access to expensive reagents and equipment. Here, we outline a simple strategy to enrich and detect anti-Neu5Gc antibodies from small volume human serological samples. This approach involves an affinity purification enrichment step, followed by an ELISA-based detection step. It exploits CMAH- transfected human cells as a source of diverse Neu5Gc-containing human glycans, with parental cells used as a matched Neu5Gc-negative control. This strategy successfully enriched Neu5Gc-specific antibodies from intravenous immunoglobulin (IVIG) and individual plasma specimens from ten healthy donors. Anti-Neu5Gc antibodies were detected in all donors. The assay was also sufficiently sensitive to observe reproducible individual differences in the anti-Neu5Gc reactivity of different donor specimens. Finally, despite this individual variation, enriched antibodies from all donor specimens bound effectively to Neu5Gc-containing glycans presented on the surface of whole human cells.