Phage Immunoprecipitation and Sequencing-a Versatile Technique for Mapping the Antibody Reactome

Abstract

Characterizing the antibody reactome for circulating antibodies provide insight into pathogen exposure, allergies, and autoimmune diseases. This is important for biomarker discovery, clinical diagnosis, and prognosis of disease progression, as well as population-level insights into the immune system. The emerging technology phage display immunoprecipitation and sequencing (PhIP-seq) is a high-throughput method for identifying antigens/epitopes of the antibody reactome. In PhIP-seq, libraries with sequences of defined lengths and overlapping segments are bioinformatically designed using naturally occurring proteins and cloned into phage genomes to be displayed on the surface. These libraries are used in immunoprecipitation experiments of circulating antibodies. This can be done with parallel samples from multiple sources, and the DNA inserts from the bound phages are barcoded and subjected to next-generation sequencing for hit determination. PhIP-seq is a powerful technique for characterizing the antibody reactome that has undergone rapid advances in recent years. In this review, we comprehensively describe the history of PhIP-seq and discuss recent advances in library design and applications.

Keywords: PhIP-seq; antigen discovery; autoimmunity; biomarker; phage display.

Graphical abstract

Fig. 1

Schematic representation of PhIP-seq. Library design and synthesis, PhIP-seq selection, next-generation sequencing, and statistical analysis. 1, the ORFs of the proteins of interest were bioinformatically extracted and segmented into peptides of equal length with an overlap to avoid splitting epitopes. 2, the peptide sequences were reverse translated into DNA to produce an oligonucleotide pool. 3, incorporate the DNA via ligation, packing, and amplification to pair particles expressing the peptides on the surface. 4, preform selection by mixing phage and serum/plasma/CSF and pull down with magnetic beads binding of the antibodies and washing away unbound phages. 5, PCR products were used to amplify and barcode the sequences, which were subsequently subjected to next-generation sequencing. Specific barcodes for demultiplexing the sequences to the right selection. 6, statistical analysis for hit identification and subsequent data analysis. CSF, cerebrospinal fluid; PhIP-Seq, phage display immunoprecipitation and sequencing.