The procedure relative to nanobody isolation and characterization was presented in a previous report . The best clone recovered after panning (E12) was subcloned into a pET14derived expression vector for the production of the nanobody fused to 6xHis and to SpyTag in E. coli . Subsequently, the construct was transformed into BL21 (DE3) SOX cells for cytoplasmic expression and purified by metal affinity chromatography, as described previously . Nanobody concentration was quantified by the Bradford method and its quality was evaluated through SDS-PAGE and gel filtration techniques.