License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: September 09, 2025
Last Modified: December 30, 2025
Collection Integer ID: 226874
Keywords: autoantibody, autoimmunity, phage display, PhIP-Seq, panning, pathogen, immunopsychiatry, immunprecipitation, T7, phage display immunoprecipitation sequencing, novel immune epitope database, profiling antibody specificity, reproducible antibody profiling, reproducible antibody profiling for researcher, broad sampling of relevant microbial antigen, sequencing library preparation, antibody specificity, throughput identification of epitope, antibodies in biological sample, translational immunopsychiatry unit, amino acid peptide, cryptic epitope, relevant microbial antigen, peptide, predicted microprotein, panseq library, antibody, peptide overlap tile design for reference, human protein, acid peptide, using commercial antibody, commercial antibody, finer epitope mapping, panseq, validation of panseq, designing panseq, antigen, existing microbial library, amino, alternative splicing, epitope, peptide overlap tile design, entire human proteome, microbial library, tiu phage display platform, phage, calibrated pcr protocol
Funders Acknowledgements:
National Institute of Mental Health
Grant ID: 1ZIAMH002982
Foundation for the National Institutes of Health
Grant ID: Deeda Blair Research Initiative for Disorders of the Brain
Abstract
The Translational Immunopsychiatry Unit (TIU) at the National Institute of Mental Health (NIMH) is engaged in autoantibody discovery and anti-pathogen antibody profiling in diverse neuropsychiatric populations.
One tool we use is Phage Display Immunoprecipitation Sequencing (PhIP-Seq), a powerful method for profiling antibody specificities, enabling high-throughput identification of epitopes recognized by antibodies in biological samples.
Our PanSeq library incorporates relatively longer, 64 amino-acid peptides, increased tiling density, a modular pool design that encompasses the entire human proteome, and a broad sampling of relevant microbial antigens. Improvements in DNA oligonucleotide synthesis have yielded a library with low intrinsic error rates.
Furthermore, our PhIP-Seq protocol implements technical replicates and a carefully calibrated PCR protocol for sequencing library preparation. We expect that these design and protocol modifications will allow for finer epitope mapping, improved detection of novel and cryptic epitopes (including those arising from alternative splicing or rare genetic variants), and fewer false positives.
In designing PanSeq, we prioritized:
A 2/3 peptide overlap tile design for reference human proteins that enhances redundancy and facilitates finer epitope mapping;
Inclusion of all predicted neoepitopes arising from single intron retention or exon exclusion splice events;
Inclusion of non-canonical autoantigens (e.g., experimentally predicted microproteins, human endogenous retroviral elements, and LINE-1 elements);
Reuse of existing microbial libraries for backwards compatibility and comparability with prior studies;
Supplementation of existing microbial libraries with a novel immune epitope database (IEDB) library
Modularity for subpool screening
In this protocol series, we detail the design, construction, and validation of PanSeq using commercial antibodies. This protocol series will serve as the primary reference for TIU publications that employ PhIP-Seq.
This protocol series is a living document that will be updated and amended as the protocol matures.
We hope these resources will facilitate robust, reproducible antibody profiling for researchers across disciplines.
Pavel KhilTranslational Immunopsychiatry Unit, NIMH
Protocol references
Larman HB, Zhao Z, Laserson U, Li MZ, Ciccia A, Gakidis MA, Church GM, Kesari S, Leproust EM, Solimini NL, Elledge SJ. Autoantigen discovery with a synthetic human peptidome. Nat Biotechnol. 2011 May 22;29(6):535-41. doi: 10.1038/nbt.1856. PMID: 21602805; PMCID: PMC4169279.
O'Donovan B, Mandel-Brehm C, Vazquez SE, Liu J, Parent AV, Anderson MS, Kassimatis T, Zekeridou A, Hauser SL, Pittock SJ, Chow E, Wilson MR, DeRisi JL. High-resolution epitope mapping of anti-Hu and anti-Yo autoimmunity by programmable phage display. Brain Commun. 2020 Aug 3;2(2):fcaa059. doi: 10.1093/braincomms/fcaa059. PMID: 32954318; PMCID: PMC7425417.
Acknowledgements
This research was supported by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH author(s) were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.
