Detection of Antigen Presentation by Murine Bone Marrow-Derived Dendritic Cells

Nanoparticles have been extensively tested as therapeutic vaccine delivery vehicles for the treatment of cancer. A key effort in this area is the delivery of antigens to antigen-presenting cells (APCs), such that tumor-specific peptides are presented in the context of class I major histocompatibility complex (MHC) molecules. The purpose of this protocol is to determine the ability of nanoparticles to deliver antigen to APCs, such that an antigenic class I peptide sequence is properly presented in the context of MHC. For the purposes of this assay, the model antigen ovalbumin (OVA) is used in the context of murine cells. The protocol requires the synthesis of nanoparticles that deliver SIINFEKL (OVA257-264), the immunodominant class I peptide derived from OVA, either in its peptide form or as part of a larger molecule (such as the whole OVA protein). The 25-D1.16 antibody, which binds to SIINFEKL presented in the context of the mouse H2-Kb MHC molecule, is used to detect antigen presentation on APCs after nanoparticle delivery. The results of this protocol may be used to infer the ability of the nanoparticle system to deliver other similar antigens.