Selection and evaluation of single domain antibodies toward MS2 phage and coat protein.

摘要

MS2 phage (MS2 ?) is a coli phage, non-pathogenic to eukaryotic cells, which has been used as a simulant for viral biothreats, such as those causing smallpox and hemorrhagic fever. MS2 ? consists of an icosahedral capsid, 28nm in diameter, and a single stranded RNA genome; the viral capsid is composed of 180 copies of coat protein (CP). In this study, we isolated anti-MS2 ? single domain antibodies (sdAbs) for the sensitive detection of the MS2 ?. To achieve this, a first immune sdAb library was prepared from llamas immunized with purified coat protein and a second from animals immunized with MS2 ?. By panning the two libraries against CP, MS2 ?, or alternating between the two targets, anti-MS2 ? and anti-CP sdAbs were selected, sequenced, and characterized for their binding affinity. Both direct binding assays and capture sandwich assays were performed on the MAGPIX platform. One of the best anti-MS2 ? sdAb, Lib2CP12H, could detect MS2 ? concentrations as low as 1.45ng/mL (～5.0E+6pfu/mL), providing equivalent detection to conventional antibodies. This sdAb is thermally stable with a melting temperature around 60°C and recovered 80% of its secondary structure after heat denaturation.