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Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments
Published on: October 15, 2016
Single-chain variable fragment (scFv) antibodies against rotavirus NSP4 enterotoxin generated by phage display
Jesús Rodríguez-Díaz1, Vicente Monedero, Gaspar Pérez-Martínez
1Department of Microbiology, School of Medicine, University of Valencia, Hospital Clinico Universitario, Avda. Blasco Ibañez 17, 46010 Valencia, Spain.
Abstract:
The rotavirus non-structural NSP4 protein causes membrane destabilization as well as an increase in intracellular calcium levels in eukaryotic cells and induces diarrhea in young mice, acting as a viral enterotoxin. In this study the phage display technique was used to generate a panel of single-chain variable fragment (scFv) antibodies specific for the NSP4 protein of the human rotavirus strain Wa from a human semi-synthetic scFv library. After several rounds of panning and selection on NSP4 adsorbed to polystyrene tubes, individual scFv were isolated and characterised by fingerprinting and by sequencing the VH and VL genes. The isolated scFv antibodies specifically recognize NSP4 in enzyme immunoassay and in Western blot. Four truncated forms of the NSP4 protein were constructed which allowed us to map the binding region of the selected scFv antibodies to the C-terminal portion of NSP4. The isolated scFv antibodies constitute valuable tools to analyse the mechanisms of NSP4 functions.
Insights
Researchers developed specific antibodies to study rotavirus NSP4 protein, a viral enterotoxin causing diarrhea. These antibodies target the C-terminal region, aiding analysis of NSP4
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Rotavirus non-structural NSP4 protein acts as a viral enterotoxin, inducing diarrhea by destabilizing cell membranes and increasing intracellular calcium.
- Understanding NSP4's function is crucial for developing rotavirus therapies.
Purpose of the Study:
- To generate and characterize single-chain variable fragment (scFv) antibodies specific for the human rotavirus Wa strain's NSP4 protein.
- To utilize these antibodies as tools for analyzing NSP4's functional mechanisms.
Main Methods:
- Phage display technique using a human semi-synthetic scFv library.
- Panning and selection of scFv antibodies against immobilized NSP4.
- Characterization of isolated scFv by fingerprinting, gene sequencing, enzyme immunoassay, and Western blot.
- Mapping of antibody binding regions using truncated NSP4 protein forms.
Main Results:
- A panel of scFv antibodies specifically recognizing the rotavirus NSP4 protein was successfully generated.
- The isolated scFv antibodies demonstrated specific binding to NSP4 in enzyme immunoassays and Western blots.
- Antibody binding was mapped to the C-terminal portion of the NSP4 protein.
Conclusions:
- The generated scFv antibodies are specific tools for detecting and studying rotavirus NSP4.
- These antibodies provide a valuable resource for further investigation into the mechanisms underlying NSP4's enterotoxic functions.
