Ever since the discovery of antibodies, they have been generated by complicated multi-step procedures. Typically, these involve sequencing, cloning, and screening after expression of the antibodies in a suitable organism and format. Here, a staphylococcal nanobody display is described that omits many the abovementioned intermediate steps and allows for simultaneous screening of multiple targets without prior knowledge nor expression of the binders. This paper reports a detailed, general step-by-step protocol to achieve nanobodies of high affinity. Apart from its focus on radioactive and fluorescent targets, it gives options for various other target formats and additional applications for the staphylococcal library; including flow cytometry and immunoprecipitation. This provides a system for antibody engineers that can be easily adopted to their specific needs.
Keywords: Bacterial surface display; Immunoprecipitation; Nanobody (VHH); Staphylococcal sortase A (SrtA); Target identification.