Cloning and Selection from Antigen Fragment Libraries for Epitope Identification

To understand what drives an immune response, it is important to characterize, at a molecular level, the site(s) on an immunogenic antigen that is directly contacted by a soluble antibody or B-cell antigen receptor (BCR) on the surface of a B lymphocyte. Moreover, antibody binding interactions with a microbial protein can interfere with the functional activity of a toxin (i.e., neutralization) and/or can aid in the clearance of the microbial protein from the body, further underscoring the importance of such characterization. Phage display technology is a potent tool that can be used to study any type of protein-protein interaction. In recent years, we have refined methods for the identification of the minimal binding contact sites of an antibody with an antigen. Here, we describe a workflow for optimizing antibody-mediated selection and for the identification and characterization of antigen-specific epitopes. This workflow includes (1) the generation of large libraries of random fragments of a gene of interest cloned into the validated pComb-Opti8 phagemid expression cloning vector system; (2) electroporation of these libraries into electrocompetent bacterial cells and subsequent recovery of viral particles, each of which displays the cloned gene fragment product as a fusion protein with the filamentous phage major coat protein VIII (pVIII); (3) recovery of individual phagemid clones that express the smallest functional epitopes recognized by an experimental antibody; (4) an efficient means of using high-throughput DNA sequencing to interrogate sequentially selected libraries to rapidly identify the gene subregions encoding epitopes of interest; and (5) means for the further characterization of potential antibody-epitope binding interactions.