69 Institutional Review Board of the UMCG (in Dutch: “Medisch Ethische Toetsingscommissie”, METc) under registration number 2008/338 and the study has been conducted in accordance with the principles of the Declaration of Helsinki (2013). Patients provided written informed consent for their participation in the study. Further details on the subcohort of 1000IBD of which PhIP-Seq profiles were generated can be found elsewhere.29 PhIP-Seq library design, preparation, sequencing and processing Library description can be found in21 (microbiota antigens) and23 (allergen databases, complete IEDB, phages). The general PhIP-Seq protocol is described in13 and was performed with minor modifications outlined by.21 In short, PCR plates in contact with phage/antibody mixtures were blocked with bovine serum albumin (BSA) solution (concentration as described in.21) BSA was supplemented into phage-buffer mixtures for immunoprecipitations (IPs). Phage wash buffer for IPs contained 0.1% (wt/vol) IPEGAL® CA 630 (Sigma-Aldrich cat. no.I3021). Phage and antibody amounts for IPs were used as optimized by21 at 3 μg of serum IgG antibodies (measured by ELISA) and phage library at 4,000-fold coverage of phages per library variant. As technical replicates of the same sample were in excellent agreement (average Pearson ρ = 0.96)21, measurements were performed in single reactions. The libraries21 (230 nt, 244,000 variants) were mixed in a 2:1 ratio with the phage, immune and allergen library (200 nt, 100,000 variants).23 Phage–antibody mixtures mixed with overhead mixing at 4°C. A 50%-50% mix of protein A and G magnetic beads (total 40 μl; Thermo Fisher Scientific, cat. nos. 10008D and 10009D, prepared according to the manufacturer’s recommendations) was added after overnight incubation and further rotated at 4°C for 4 h, then the beads were transferred to PCR plates and washed twice, as previously reported.21 Therefore, a Tecan Freedom Evo liquid-handling robot with filter tips was used. PCR amplifications (pooled Illumina amplicon sequencing) were run with Q5 polymerase (New England Biolabs, cat. no. M0493L) according to the manufacturer’s recommendations (primer pairs as outlined by21). Composition of the antigen library This work uses two previously developed peptide libraries: a microbial library21 and an allergen library.23 The microbial library contains 244,000 peptide sequences from 28,668 different proteins, from which 27,837 proteins were derived frommicrobial antigens, while the rest are controls. This contains genes predicted from metagenome assembled genomes (147,061 peptides), known pathogenic bacterial species (61,250 peptides), bacteria known to be coated with antibodies (22,050 peptides), probiotic bacteria (14,700 peptides), virulence factors extracted from the virulence factor database (VFDB) (24,164 peptides) and controls (11,525 oligos). Antigens were selected giving priority to known immunogenic antigens and focusing on secreted, membrane and motility proteins. The second library contained 5,527 peptides from five different allergen databases,23 31,436 peptides from the Immune Epitope Database (IEDB)24 and approximately 40,000 phage peptides. Determinants of the human antibody epitope repertoire
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