Selection and evaluation of single domain antibodies toward MS2 phage and coat protein.
MS2 phage (MS2 Ø) 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 Ø 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 Ø single domain antibodies (sdAbs) for the sensitive detection of the MS2 Ø. 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 Ø. By panning the two libraries against CP, MS2 Ø, or alternating between the two targets, anti-MS2 Ø 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 Ø sdAb, Lib2CP12H, could detect MS2 Ø 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.
Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, 4555 Overlook Ave. SW, Washington, DC 20375, USA. jinny.liu@nrl.navy.mil
Jinny L
JL
Liu
Daniel
D
Zabetakis
Ellen R
ER
Goldman
George P
GP
Anderson
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology* / Q000302:isolation & purification*; D000918:Antibody Specificity; D002161:Camelids, New World; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology*; D015723:Gene Library; D017909:Levivirus / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data
D000914:Antibodies, Viral; D036022:Capsid Proteins