In vitro generation of rabbit anti-Listeria monocytogenes monoclonal antibody using single cell based RT-PCR linked cell-free expression systems.

Rabbit monoclonal antibodies (mAbs) have advantages over mouse antibodies in diagnostics and biotechnological applications owing to higher affinity and specificity.We developed a platform to generate rabbit mAbs by a novel monoclonal antibody generation method named “Single-Cell Reverse Transcription-PCR linked in vitro-Expression (SICREX)” system. In this method,we use single-cell based RT-PCR followed by sequential PCR steps of mAb genes and subsequent cell-free protein synthesis (CFPS) by using linear DNA fragments of mAbs. This platform enables the rapid generation and evaluation of mAbs derived from antigen-specific single B cells in the peripheral blood of immunized animals without mammalian cell cultivation. In this study, the antigen used was a food-borne gram-positive pathogen, Listeria monocytogenes, that is known to cause serious infection. Three active mAbs in CFPS were obtained by constructing the single chain of variable fragment (scFv) form. These scFvs were produced in the cytoplasm of E. coli Shuffle T7 Express strain as an active form and used for further investigation.