Identification of immunogenic commensal antigens using phage display

Abstract

Humoral immunity plays a major role in the establishment and maintenance of host-microbiota commensalism and immunity to pathogenic microorganisms. However, identification of antigens eliciting adaptive immune responses within barrier and systemic tissues represents a significant hurdle to further understanding this host-microbe dialogue. Here, we provide a protocol to identify immunogenic protein antigens expressed by commensal and pathogenic microbes by using bacteriophage (phage) display-mediated antibody/antigen biopanning. The procedure entails generation of an M13-phagemid library, production of an ORFeome (totality of open reading frames) phage library followed by multiple rounds of affinity-based immunoprecipitation and subsequent antigen validation by ELISA, ELISPOT and/or B cell tetramer generation. The protocol is optimized to identify antigens eliciting both IgA and IgG isotype responses and can use either circulating or intestinal antibodies for antigen identification. Generation and isolation of monoclonal phage encoding putative protein antigens enable simple identification of immunogenic antigens by Sanger sequencing, often providing protein domain-level resolution of epitope-bearing regions. Our protocol can be carried out by a trained molecular biologist and enables antigen identification and validation in the timeframe of weeks.