A method for rapid nanobody screening with no bias of the library diversity

Abstract

AbstractNanobody refers to the variable domain of heavy-chain-only antibodies. The distinctive advantages of nanobodies including small size, feasible expression inEscherichia 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. Here, based on the unique properties of nanobodies, we developed an integrated method to screen and express nanobodies simultaneously with no bias of the library diversity. The library of nanobodies was cloned and secretively expressed into the culture medium. Target specifical binding nanobodies were isolated through 1-3 rounds of dilution 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 more than 10 folds upon each round of dilution. Ultimately, 5 nanobodies against the death domain receptor 5 (DR5) and 5 nanobodies against thePyrococcus furiosus(Pfu) DNA polymerase were produced directly out of their immunized libraries, respectively. Additionally, our approach allowed nanobody screening even without any specialized instruments/devices, demonstrating general applicability in the routine production of monoclonal nanobodies for diverse biomedical applications.