51 Figure 2 | Conventional and state-of-the-art antibody profiling technologies. Schematic overview of the key experimental principles underlying the respective methodologies. More details on their general use and application in IBD can be found inTable 1 (ELISA) and elsewhere (peptide array:3, PhIP-Seq:4,38,52-55, IgA-Seq:56-59, BCR-Seq:60-65). Abbreviations: BCR-Seq, B cell receptor sequencing; CD, Crohn’s disease; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; IgA-Seq, immunoglobulin A sequencing; PhIP-Seq, phage-display immunoprecipitation sequencing; PTMs, posttranslational modifications; SHM, somatic hypermutation; SLE, systemic lupus erythematosus; UC, ulcerative colitis. However, in IBD, a large number of antigens with diagnostic potential have already been reported (Table 1). Furthermore, given the large number of different bacterial species making up the human microbiome51 and increased bacterial translocation in IBD, many additional microbial antigens could be targeted by antibody responses. Alexander et al.3 applied a protein array (Figure 2) to tap into this uncharacterized space of potential IBD biomarkers. In contrast to ELISAs, arraybased technologies allow spotting of hundreds to thousands of antigens on carrier surfaces. Antibody binding against all antigens can be assessed in parallel using optical fluorescence readouts. Using such a protein array, Alexander et al. detected significantly increased antibody responses against Lachnospiraceae flagellins in patients with CD compared to patients with UC or healthy controls. While anti-flagellin Ig responses in IBD were already known (Table 1), this protein array study enabled the researchers to narrow down the exact bacterial species targeted. Despite accelerating detection by parallelized readouts, protein arrays still require the laborious production of every antigen to be tested, typically making studies on more than a few thousand antigens unfeasible. Phage-display immunoprecipitation sequencing (PhIP-Seq) is a methodology that circumvents this lengthy step of antigen production.52 PhIP-Seq antigens are encoded by synthetic DNA oligos released frommicroarray slides, allowing the generation of hundreds of thousands of variants in parallel. The sequences of the DNA oligos can be selected fromvirtually any source, such as databases or metagenomic sequencing. To date, PhIP-Seq libraries have been generated for the human proteome53, viruses54,55 and the gut microbiota.4 The synthetic DNA oligoes are cloned into the genome of T7 phages and translated into peptide antigens that are subsequently displayed as fusion to a phage surface protein. Antibody binding is assessed by next generation sequencing (NGS), as every phage contains the DNA encoding the protein on its surface, DNA reads of bound antigens are increased after washing away unbound phages in an immunoprecipitation step.52 For studying Ig epitope repertoires against the gut microbiota, various strategies4 have been applied to enrich for potential antigens that are bound by antibodies (as the coding capacity of bacterial microbiota is enormous51 and would exceed current library sizes achievable by PhIPSeq). Existing microbiota antigen libraries have focused on secreted-, membrane-, and surfaceexposed proteins and virulence factors, relying on bioinformatic strategies to annotate protein functions.4 This rational selection of potential antigens to be presented by the phages is a key advantage compared to ‘traditional’ phage display, where antigens are randomly cloned from metagenomic DNA libraries. Cloning of metagenomic DNA by restriction digestion also leads to truncated gene sequences, frameshifts, genes inserted in the wrong orientation, as well as Antibody signatures in IBD: developments and applications
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