Selection and evaluation of single domain antibodies toward MS2 phage and coat protein
Jinny L Liu1, Daniel Zabetakis, Ellen R Goldman
1Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, 4555 Overlook Ave. SW, Washington, DC 20375, USA. jinny.liu@nrl.navy.mil
Abstract:
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.
Insights
Researchers developed novel single domain antibodies (sdAbs) for sensitive MS2 phage detection. One sdAb, Lib2CP12H, achieved detection limits comparable to conventional antibodies, showing high thermal stability.
Area of Science:
- Biotechnology
- Virology
- Immunology
Background:
- MS2 phage (MS2 Ø) serves as a simulant for dangerous viral biothreats.
- MS2 Ø has an icosahedral capsid (28nm) composed of coat protein (CP) and a single-stranded RNA genome.
- Sensitive detection methods are crucial for biodefense applications.
Purpose of the Study:
- To isolate and characterize single domain antibodies (sdAbs) for sensitive MS2 phage detection.
- To evaluate the performance of these sdAbs using various assay formats.
- To identify a highly stable and effective sdAb for MS2 Ø detection.
Main Methods:
- Generation of llama immune sdAb libraries against MS2 phage coat protein (CP) and whole MS2 Ø.
- Panning of libraries against CP and MS2 Ø to select anti-MS2 Ø and anti-CP sdAbs.
- Characterization of sdAb binding affinity using direct binding and capture sandwich assays on the MAGPIX platform.
Main Results:
- Selected anti-MS2 Ø sdAbs demonstrated high binding affinity and sensitive detection capabilities.
- One lead sdAb, Lib2CP12H, detected MS2 Ø at concentrations as low as 1.45 ng/mL (∼5.0E+6 pfu/mL).
- Lib2CP12H exhibited excellent thermal stability (Tm ≈ 60°C) and structural recovery after heat denaturation.
Conclusions:
- Single domain antibodies are effective tools for sensitive MS2 phage detection.
- The identified sdAb, Lib2CP12H, offers comparable performance to conventional antibodies with enhanced stability.
- These findings support the development of advanced diagnostic tools for viral simulants and potential biothreat agents.
