Verma Sheenam, Kimmel Samantha, Harrison Oliver J
Center for Fundamental Immunology, Benaroya Research Institute, Seattle, WA, USA.
Department of Pediatrics, University of California San Diego, La Jolla, CA, USA.
Nat Protoc. 2025 Jun 17. doi: 10.1038/s41596-025-01193-1.
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.
体液免疫在宿主-微生物共生关系的建立和维持以及对病原微生物的免疫中发挥着重要作用。然而，在屏障组织和全身组织中鉴定引发适应性免疫反应的抗原是进一步理解这种宿主-微生物对话的一个重大障碍。在此，我们提供了一种通过使用噬菌体展示介导的抗体/抗原生物淘选来鉴定共生微生物和病原微生物表达的免疫原性蛋白质抗原的方案。该程序包括生成一个M13噬菌粒文库，构建一个开放阅读框（ORF）噬菌体文库，随后进行多轮基于亲和力的免疫沉淀，以及通过酶联免疫吸附测定（ELISA）、酶联免疫斑点分析（ELISPOT）和/或B细胞四聚体生成进行后续抗原验证。该方案经过优化，可鉴定引发IgA和IgG同种型反应的抗原，并且可以使用循环抗体或肠道抗体进行抗原鉴定。生成和分离编码推定蛋白质抗原的单克隆噬菌体能够通过桑格测序简单鉴定免疫原性抗原，通常可提供表位区域的蛋白质结构域水平分辨率。我们的方案可由训练有素的分子生物学家实施，并能在数周内完成抗原鉴定和验证。
