A robust method for the rapid generation of nanobodies

Background: ： Nanobody refers to the variable domain of heavy-chain only antibodies. The distinctive advantages of nanobodies including small size, feasible expression in Escherichia coli ( E. coli ), and superior stability make them promising tools for applications in scientific research and therapies. So far, the screening and expression of nanobodies are mainly following similar methods used for conventional antibodies, suffering from amplification-caused losses of the diversity of libraries and requirements of subcloning of interests into the expression vector. Results: : Here, based on the unique properties of nanobodies, we developed a new high-throughput (>10 5 ) method to screen and express nanobodies simultaneously in the cell culture media, simplifying the production of specific nanobodies significantly. Nanobodies specifically binding with target proteins were enriched through cell splitting and regrown steps in a way following the Poisson distribution to ensure no positive clones were dismissed, while the population of positive clones increased by one order of magnitude upon each round of cell splitting. In the end, 5 different nanobodies against the death domain receptor 5 (DR5) and 5 different nanobodies against the Pyrococcus furiosus ( Pfu ) DNA polymerase were produced directly out of their immunized libraries respectively. Conclusions: : Our approach offers an integrated strategy for rapid, and low-cost nanobody screening and expression with no loss of the library diversity, demonstrating general applicability in the routine production of monoclonal nanobodies for diverse biomedical applications.