Synthetic High‐Throughput Microarrays of Peptidoglycan Fragments as a Novel Sero‐Diagnostic Tool for Patient Antibody Profiling

Peptidoglycan (PGN) is a complex biopolymer crucial for cell wall integrity and function of all bacterial species. While the strong inflammatory properties of PGN and its derived muropeptides are well‐documented in human innate immune responses, adaptive immunity, including antibody responses to PGN, remain inadequately characterized. Microarray technology represents a cost‐ and time‐efficient method for studying such interactions. Our laser‐based technology enables the high‐throughput synthesis of biomolecules on functionalized glass slides. Here, this on‐chip synthesis was developed for PGN fragments, to generate a variety of 216 stem peptides and attach six different glycan moieties that are major structural components of bacterial cell walls. Thereby, 864 PGN fragments from different Gram‐negative and Gram‐positive species were generated. The arrays were validated with four different monoclonal antibodies against PGN or poly‐N‐acetyl glucosamine and identified their epitopes. Finally, proof of concept for antibody profiling in patient samples was performed by comparing a panel of well‐characterized plasma samples of epidermolysis bullosa (EB) patients suffering from (chronic) wounds with Staphylococcus aureus infection. EB patients show an increased response to the muramyl dipeptide. Therefore, this novel high‐throughput PGN glycopeptide microarray technology promises to identify distinct antibody profiles against human microbiomes in diseases, notably in those involving the intestine.