Cloning and Selection from Antigen Fragment Libraries for Epitope Identification

This is the authors' abstract. We don't add key points for this paper.

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.